(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property
Organization
International Bureau
(43) International Publication Date
29 December 2004 (29.12.2004)
PCT
llll II III I II II II I II III Mill Mil II III
(10) International Publication Number
WO 2004/113492 Al
(51) International Patent Classification 7 : C12M 3/00,
C12N 5/00
(74) Agent: G.E. EHRLICH (1995) LTD.; 11 Menachem Be
gin Street, 52 521 Ramat Gan (IL).
(21) International Application Number:
PCT/IL2004/000571
(22) International Filing Date: 27 June 2004 (27.06.2004)
(25) Filing Language: English
(26) Publication Language:
English
(30)
Priority Data:
60/482,437
60/488,408
60/517,073
60/517,084
60/544,356
60/544,357
26 June 2003 (26.06.2003) US
21 July 2003 (21.07.2003) US
5 November 2003 (05.1 1.2003) US
5 November 2003 (05.1 1.2003) US
17 February 2004 (17.02.2004) US
17 February 2004 (17.02.2004) US
(71) Applicant (for all designated States except US ) : MOLEC-
ULAR CYTOMICS LTD. [CY/CY]; P.O. Box 21255,
Nicosia, 1505 (CY).
(72) Inventors; and
(75) Inventors/Applicants (for US only): DEUTSCH,
Mordechai [IL/IL]; 73 Moshav Olesh, 42 855 Doar Na
Lev-HaSharon (IL). HERZBERG, Max [IL/IL]; Moshav
Sitria, P. O. Box 7, 76 834 Emeq Soreq (IL). TIROSH,
Reuven [IL/IL]; 20 Ben Gurion Street, 44257 Kfar Saba
(IL). DEUTSCH, Assaf [IL/IL]; 12 Moshav Tzofaria, 60
932 Moshav Tzofaria (IL).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FT,
GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI,
SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
GW, ML, MR, NE, SN, TD, TG).
Published:
— with international search report
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
OS
o
(54) Title: IMPROVED MATERIALS FOR CONSTRUCTING CELL-CHIPS, CELL-CHIP COVERS, CELL-CHIP COATS, PRO-
CESSED CELL-CHIPS AND USES THEREOF
(57) Abstract: A chip-device is provided for holding living cells in a plurality of wells disposed on the surface of a carrier, char-
acterized in that the wells are configures to influence the proliferation of living cells in the wells, for example by changing the size
of the wells, delaying or inhibiting cell -proliferation, delaying or inhibiting cell adhesion, or allowing cell proliferation inside or
through components of the device itself. The influence on cell proliferation is achieved, for example, by coating the inside of the
wells or fashioning the carrier from materials such as gels, especially hydrogels, polydimethylsiloxane, elastomers or polymerized
para-xylylene molecules. Provided is also a gel-cover for any cell-holding device. Provided is also a method for making the devices
of the present invention. Provided is also a method of manipulating cells by covering wells where the cells are held with gel or by
increasing the size of wells wherein cells are held. Provided is also a method of collecting cells from a biological sample onto a
well-bearing device.
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IMPROVED MATERIALS FOR CONSTRUCTING CELL-CHIPS, CELL-CHIP
COVERS, CELL-CHIP COATS, PROCESSED CELL-CHIPS AND USES THEREOF
5 FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to the field of cellular biology and more
particularly, to an improved device for the study of cells as well as a method for
producing the device. The present invention is also of methods for the study of cells, the
methods implementable using devices of the present invention.
10 Combinatorial methods in chemistry, cellular biology and biochemistry are
essential for the preparation of multitudes of active entities such as molecules. Once
such active entities are prepared, it is necessary to study the effect of each of the active
entities on living organisms. The study of the effects of active entities on living
organisms is often performed on living cells. Cell-functions include many interrelated
15 pathways, cycles and chemical reactions. Often, a study of an aggregate of cells,
whether homogenous or heterogenous, does not provide interpretable results. Thus the
comprehensive study of the effects of an active entity may require the examination of
the effect of the active entity of single isolated living cells. Thus, the use of single-cell
assays is one of the most important tools for understanding biological systems and the
20 influence thereupon by various stimuli.
The combinatorial preparation of multitudes of active entities coupled with the
necessity of studying the effects of all the active entitities using live-cell assays, requires
the development of high-throughput methods for studying living cells, especially single
live-cell assays.
25 In the art, various different methods for studying living cells are known.
Multiwell microtiter plates having 6, 12, 48, 96, 384 or even 1536 wells on a
standard ca. 8.5 cm by ca. 12.5 cm footprint are well known in the art. The volume of
the wells depends on the number of wells and the depth thereof but generally is greater
than 5 x 10' 6 liter (for a 1536 well plate). Although exceptionally useful for the study of
30 large groups of cells, multiwell microtiter plates are not suitable for the study of
individual cells or even small groups of cells due to the large, relative to the cellular
scale, size of the wells. Generally, cells held in such wells float about a solution in the
wells and are not easily found for observation. When cells adhere to a well surface, the
cells adhere to any location in the well, including anywhere on the bottom of the well
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and on the walls of the well. Such variability in location makes high throughput imaging
(for example for morphological studies) challenging as acquiring an individual cell and
focussing thereon is extremely difficult. Such variability in location also makes high-
throughput signal processing (for example, detection of light emitted by a single cell
5 through fluorescent processes) challenging as light must be gathered from the entire
area of the well, increasing the noise in the signal. Further, cells held inside a large well
of a microtiter plate can be physically manipulated only with difficulty. Thus, multiwell
microtiter plates are in general only suitable for the study of large numbers of cells as a
group.
10 In the art, a number of method and devices have been developed for the study of
individual cells or a small number of cells as a group. Many such methods are based on
using well-bearing devices. A well-bearing device is a device for the study of cells that
has at least one well-bearing component for study of cells. A well-bearing component is
a component having at least one, but generally a plurality of wells, each well configured
15 to hold at least one cell. The term "well" is quite general and includes such features as
dimples, depressions, tubes and enclosures. Since cells range in size from about 1
microns to about 100 (or even more) microns diameter there is no single well size that is
appropriate for holding one cell of any type. That said, the dimensions of the typical
individual well in the well-bearing components known in the art have dimensions of
20 between about 1 microns up to about 200 microns, depending on the exact
implementation. For example, a device designed for the study of single isolated 20
micron cells typically has wells of dimensions of about 20 microns. In other cases,
larger wells are used to study the interactions of a few cells held together in one well.
For example, a 200 micron well is recognized as being useful for the study of the
25 interactions of two or three cells, see PCT patent application IL0 1/00992 published as
WO 03/035824.
One feature that increases the utility of a well-bearing device is that each
individual well is individually addressable. By individual adressability is meant that
each well registered, found or studied without continuous observation. For example,
30 after cells are held in wells of a well-bearing component, each cell is characterized and
the respective well where each cell is held is noted. When desired, the observation
component of the well-bearing device is directed to the location of the well where a
specific cell is held. One method of implementing individual adressability is by the use
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of fiducial points or other features, generally on the well-bearing component. Another
method of implementing individual adressability is by arranging the wells in a matrix of
wells and finding a desired well by counting. Another method of implementing
individual adressability is by providing a dedicated observation component for each
5 well.
In the art, the well-bearing component of well-bearing devices is often a chip, a
plate or other substantially planar component. Herein such a component is termed a
"carrier". In the art, there also exist non-carrier well-bearing components of well-
bearing devices, for example, bundles of fibers or bundles of tubes.
10 Mrksich and Whitesides, Ann. Rev. Biophys. Biomol Struct 1996, 25, 55-78;
Craighead et al, J. Vac. ScL Technol 1982, 20, 316; Singhvi et al, Science 1994, 264,
696-698; Aplin and Hughes, Analyt. Biochem. 1981, 113, 144-148 and U.S. Patent
5,324,591 all teach of devices including matrices of spots of cell-attracting or cell-
binding entities on a plate. In such devices, the spots serve as wells, binding to cells
15 through a variety of chemical bonds. In such devices, the plate is the well-bearing
component of the device. Due to the size of the spots, each such well generally holds
more than one cell To reduce interaction between cells held at different wells, the spots
must be spaced relatively far apart, reducing loading as expressed in terms of wells per
unit area. Even with wide spacing of wells, in such devices, cells are not entirely
20 isolated from mutual interaction, nor can cells be subject to individual manipulation.
The fact that the cells are not free-floating but -arc bound to the plate through some
interaction necessarily compromises the results of experiment^ performed.
In U.S. Patent 6,103,479, the well-bearing component is a transparent carrier
provided with a non-uniform pattern of wells, each well functionalized with chemical
25 entities that bind to cells specifically or non-specifically. Each well is of approximately
200 to 1000 micron diameter and holds a plurality of cells. The interwell areas are
hydrophobic so as not to attract cells. In addition to the carrier, a device of U.S. Patent
6,103,479 is provided with a chamber-bearing plate that mates with the carrier made of
glass, plastic or silicon in which individually adressable micro fluidic channels are
30 etched. When brought together, the carrier and chamber-bearing plate constitute a
casette in which each cell is bound to the carrier and isolated in a chamber provided
with a fluid delivery system. Reagents are provided through the fluid delivery system
and observed by the detection of fluoresence. In order to provide space for the walls of
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the chambers, the interwell areas of the carrier are relatively large, reducing loading as
expressed in terms of wells per unit area. Subsequent to study, the cassette is separated
into the two parts and the micro-patterned array of cells processed further. In some
embodiments, the chamber-bearing plate is made of polytetrafluoroethylene,
5 polydimethylsiloxane or an elastomer. As held cells do not make contact with the
chamber-bearing plate it is not clear what advantages are to be had when providing a
chamber-bearing plate of such esoteric materials.
In U.S. Patent 4,729,949 is taught a device for trapping individual cells in a
well-bearing carrier, the carrier being substantially a plate having a plurality of
10 individually adressable tapered apertures of a size to hold individual cells. Suction
applied from the bottom surface of the plate where the apertures are narrow creates a
force that draws cells suspended in a fluid above the carrier into the wide part of the
apertures on the surface of the carrier to be held therein. Using the teachings of U.S.
Patent 4,729,949 a specific group of cells (having dimensions similar to that of the
15 aperture) can be selected from amongst a group of cells and held in the carrier.
Although the cells are subjected to common stimuli, the fact that the wells are
individually adressable allows the effect of a stimulus on an individual cell to be
observed. A carrier of U.S. Patent 4,729,949 is generally made of metal and prepared
using standard photoresist and electroplating techniques. In a carrier of U.S. Patent
20 4,729,949, the interwell areas of the carrier are relatively large, leading to a low loading
as expressed in terms of wells per unit area. Further, the suction required to hold cells in
wells of a carrier of U.S„ Patent 4,729,949 caused deformation of a held cell and makes
a significant portion of the cell membranes unavailable for contact, both factors that
potentially compromise experimental results. Study of cells with non-fluorescence
25 based methods generally gives poor results due to reflections of light from the carrier.
In PCX patent application US99/04473 published as WO 99/45357 is taught a
well-bearing device produced by etching the ends of a bundle of optical fibers
(apparently of glass) to form a well-bearing component that is a bundle of fibers. The
size of the hexagonal wells are demonstrated to be as small as 7 micron wide, 5 micron
30 deep and have a volume of 1.45 x 10** 13 liter. The interwell area is quite significant due
to the thickness of the cladding of the optical fibers. Cells held in each well are
independently observable through a respective fiber or by observation from above. In
some embodiments, the inside surface of the wells is coated with a film of materials
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such as collagen, fibronectin, polylysine, polyethylene glycol, polystyrene,
fluorophores, chromophores, dyes or a metal. Loading the well-bearing component of
PCT patent application US99/04473 includes dipping the optical fiber bundle in a cell
suspension so that cells adhere to the wells. There are a number of disadvantages to the
5 teachings of PCT patent application US99/04473. The fact that the cells are studied only
subsequent to adhesion to the wells necessarily influences the results of experiments
performed. As cell proliferation starts soon after adhesion, it is never clear if a signal
detected results from a single cell or a plurality of cells. It is is not clear where exactly
in a well a cell is held and therefore what percentage of light emitted from a cell travels
10 to a detector. The fact that emitted light travels through an optical fiber leads to loss of
time dependent and phase information.
In PCT patent application IL04/000192 is taught a well-bearing device produced
by bundling together glass capillaries, each glass capillary attached to an independent
fluid flow generator such as a pump. A cell held in a first well is transferred to a second
15 well by the simultaneous application of an outwards flow from the first well and an
inwards flow into the second well.
A preferred device for the study of cells is described in PCT patent application
IL0 1/000992 published as WO 03/035824. The device 10, depicted in Figure 1, is
provided with a transparent carrier 12 as a well-bearing component. Carrier 12 is
20 substantially a sheet of transparent material (preferably glass or polystyrene) on the
surface of which features such as inlet connectors 14, fluid channels 16, wells (in Figure
1 a matrix of wells 18), a fluid reservoir 20, and an outlet connector 22. Carrier 12 is
immoveably held in a holder 24 having a cutout window of a size and shape to accept
carrier 12. Other components of device 10 not depicted include flow generators,
25 observation components, external tubings and the like. When a cover slip (not depicted)
is placed or integrally formed with over carrier 12, fluid channels 16, matrix of wells 18
and reservoir 20 are sealed forming channels that allow transport of fluids and reagents
to cells held in matrix of wells 18. The wells are configured, to hold one or more cells
and are preferably individually adressable both for examination and manipulation.
30 Figure 2 is a reproduction of a photograph of a different carrier 26 held in a
holder 24. A first syringe 28 as an inlet flow generator is in communication with an inlet
connector 14 by a capillary tube 30, Inlet connector 14 is in communication with matrix
of wells 18 through a fluid passage 16. Matrix of wells 18 is in communication with
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outlet connector 22 through a fluid passage 16. A second syringe 32 as an outlet flow
generator is in communication with outlet connector 22 through capillary tube 34.
PCT patent application IL0 1/000992 also teaches methods of physically
manipulating cells held in a well-bearing device, using for example, individually
5 addressable microelectrodes (found in the wells or in the cover slip) or optical tweezers.
Typical physical manipulations include moving cells into or out of wells. One useful
method that is implemented using a device of PCT patent application IL0 1/000992 is
that cells, each held alone in a respective well, are examined (either in the presence or
absence of reagents) and based on the results of the examination, cells with a certain
10 characteristic are selected to remain in a respective well while cells without the certain
characteristic are removed from a respective well and ejected by the application of a
flow in parallel to the surface of the carrier, generated by a flow generator.
An additional feature of the teachings of PCT patent application IL0 1/000992 is
that, in some embodiments, the area occupied by a well matrix is substantially entirely
15 made up of wells with little or no interwell area, see Figure 3. Figure 3 is a
reproduction of a photograph of part of a well matrix 18 from the top of a carrier 12 of
PCT patent application IL01/00992. In Figure 3 is seen a plurality of hexagonal wells
36, some populated with living cells 38, It is seen that the interwell areas 40 make up
only a minor percentage of the total area of well matrix 18. This feature allows dense
20 (near tissue density) cell packing, especially in single-cell well configurations and also
allows simple well loading: a fluid containing suspended cells is introduced in the
volume above the wells. Since there is little interwell area, cells invariably settle in
wells.
One problem of the devices known in the art is that the materials from which the
25 well-bearing components are made interfere with the study of cells. For example, in
PCT patent application EL0 1/000992 the carrier is made of a transparent material such
as glass or polystyrene. This is an excellent solution when studying cells by fluoresence.
However, despite the transparency of the carrier, the fact that the index of refraction (n)
of glass or polystyrene (n ~ 1.5) is significantly greater that that of water or the
30 physiological medium (n - 1.33) in which living cells are found leads to scattering,
reflection and diffraction of light, interfering with direct optical study of cells held in
such carriers, for example, during morphological studies using a microscope. It would
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be advantageous to have a carrier of a material that is devoid of the problems associated
with scattering, reflection and diffraction of light.
A further problem of the devices known in the art is that of proliferation of cells
held or isolated in well-bearing components. Cells are held in wells. Movement of the
5 well-bearing component causes cells to move out or be jostled from a well, leading to
cell-loss or to cell-identity loss. Since proliferation takes time, this means that an entire
device must be dedicated to studying one well-bearing component as long as there is
interest in the cells held in the well-bearing component. This problem is solved in PCT
patent application US99/04473 by encouraging cell-adhesion, but is suitable only for
10 cells that are exceptionally adhesive and even then there is no guarantee that cells will
not be lost. It has already been noted that in devices where cells are bound to wells, the
actual binding may compromise experimental results. Further, there is often a desire to
move or transport a cell-populated well-bearing component before cell adhesion has
commenced. Further, even if the well-bearing component is not moved, proliferation of
15 cells inside a well or an enclosure leads to unnatural population shapes, cell distortion
and overcrowding effects. Further, if the cell populations grow outwards from an
enclosure, the cells are subject to flow-induced loss or migration from the population
itself to contaminate other wells. It would be advantageous to have a means that allows
a cell-populated well-bearing component to be moved without concern that cells will
20 exit respective wells. Such a means would preferably allow isolation, characterization,
selection, proliferation and study of cells and at the same time allow storage, incubation
and even transport of cells held therein without identity loss. Further, it would be
advantageous to have a simple and efficient means to provide a plurality of cells that
have been selected with enough space to proliferate without overcrowding effects.
25 A further problem of devices known in the art is that of fast cell proliferation. In
some cases, cells held in wells proliferate quickly, before there is time to characterize
the cells as individuals. This is a problem that often occurs when cells are first loaded
onto a well-bearing component and stored with the intent of study at a later date, for
example when a plurality of individual cells is held in a matrix wells for use as a
30 biosensor or screening device. It would be advantageous to have a means that allows the
study of cells using a well-bearing component such as is known in the field of cellular
biology where cells are prevented, or at least delayed, from proliferating.
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A further problem of devices known in the art is that of loading devices with
cells from a sample. Generally, a sample of cells is suspended in a fluid and brought in
proximity of the well-bearing component of a given device used to study the cells. One
method of bringing the cell suspension in proximity of the well-bearing component is
5 through the fluid channels. For example, in a device of Figure 1, a cell suspension is
injected above well matrix 18 using first syringe 30. Alternatively, the cover slip is
removed, and a drop of cell suspension applied directly onto well matrix 18 and then the
cover slip put back in place. By generating a force (e.g., by activating microelectrodes)
to push or pull cells into wells or by allowing cells to settle by the force of gravity, cells
10 populate the individual wells of well matrix 18. Although such a loading method is
suitable for cell suspensions such as blood or lymphatic fluid, when it is desired to study
cells found in a solid matrix such as bodily tissue or an organ, the method if far from
ideal. The step of releasing cells from a solid matrix is a time consuming and delicate
operation. Often cells are killed by the process of release from the solid matrix. It would
15 be advantageous to have means to harvest viable cells from a solid matrix for study
using a well-bearing device.
It would be highly advantageous to have a device and methods for the study of
cells not having at least some of the disadvantages of the prior art.
20 SUMMARY OF THE INVENTION
The present invention successfully addresses at least some of the shortcomings
of the prior art by providing a new device, a method for producing the device, new
methods for studying cells, and a new method for loading the well-bearing component
of a well-bearing device.
25 According to the teachings of the present invention there is provided a
chip-device for holding living cells, the device comprising a carrier having a plurality of
wells disposed on a surface, each well configured to hold at least one (and preferably no
more than one) living cell of a certain type, the device characterized in that the wells are
configured to influence (preferably in a predetermined manner) the proliferation of
30 living cells held in the wells. It is preferred that each of the wells is individually
addressable. Each well has an "inside", that is a physical surface with which a cell held
in the well may incidentally make contact.
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In a preferred embodiment, the inside of the wells (with which held cells
incidentally make contact) comprises a material selected from the group consisting of a
gel, a hydrogel, polydimethylsiloxane, an elastomer, polymerized para-xylylene
molecules, polymerized derivatives of para-xylylene molecules and silicon rubber,
5 In a preferred embodiment, the carrier is substantially made of a material
selected from the group consisting of a gel, a hydrogel, polydimethylsiloxane, an
elastomer and silicon rubber.
Typically configured devices of the present invention have at least one feature
from amongst the six features:
10 (a) the inside of the wells is configured to delay cell proliferation;
(b) the inside of the wells is configured to inhibit cell proliferation;
(c) the wells are configured to allow cell proliferation inside and into at least one
component of the chip-device;
(e) the inside of the wells is configured to delay adhesion of living cells thereto;
15 (f) the inside of the wells is configured to inhibit adhesion of living cells thereto;
and
(g) the size of the wells is changeable.
In an embodiment of the present invention, the size of the wells of the carrier is
changeable. In such a case, generally the carrier is configured to be deformable in at
20 least one dimension and upon deformation the size of at least one of the wells is
changed, generally increased. For example, upon deformation one, two or three
dimensions of the carrier are changed, for example, the depth, the breadth, the length or
a combination of any two or three of the dimensions.
In an embodiment of the present invention, the carrier is elastically deformable.
25 Suitable materials from which to make an elastically deformable carrier include but are
not limited to elastomers, rubbers and silicon rubbers.
In an embodiment of the present invention, the carrier is plastically deformable.
Suitable materials from which to make an plastically deformable carrier include but are
not limited to hydrocarbon wax, crystalline wax, polypropylene, isotactic polypropylene
30 homopolymer, syndiotactic polypropylene homopolymer, metallocene catalyzed
isotactic polypropylene homopolymer, metallocene catalyzed syndiotactic
polypropylene homopolymer, ethylene-propylene random copolymer, butene-propylene
random copolymer, ethylene-propylene-butene-1 terpolymer, low density polyethylene,
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linear low density polyethylene, very low density polyethylene, metallocene catalyzed
polyethylene, metallocene catalyzed polyethylene copolymers, ethylene-methacrylate
copolymers, ethylene-vinyl acetate copolymers, ionomer resins, an ethylene-propylene
random copolymer, ethylene-butene-1 copolymer, ethylene-propylene-butene-1
5 terpolymer, propylene-butene copolymer, low density polyethylene, linear low density
polyethylene, very low density polyethylene, metallocene catalyzed polyethylene
plastomer, metallocene catalyzed polyethylene, metallocene catalyzed polyethylene
copolymers, ethylene-methacrylate copolymer, ethylene vinyl acetate copolymer,
ionomer resin and combinations thereof.
10 In an embodiment of the chip-device of the present invention the carrier is made
of a material having an index of refraction similar to that of water. In a preferred
embodiment of the present invention, the index of refraction of the carrier is less than
about 1.4, less than about 1.38, less than about 136, less than about 1.35, less than
about 1.34 or substantially equal to that of water.
15 In an embodiment of the chip-device of the present invention, at least one
component of the device (preferably the carrier, a cover for the well-bearing surface of
the carrier or both) is made of a gel. The component is preferably made of a transparent
gel, preferably a hydrogeL Herein, by a "transparent material" or a "transparent gel" is
meant that the material or gel is substantially transparent to wavelength regions of the
20 visible light spectrum, the ultraviolet light spectrum and/or of infrared radiation,
preferably the visible light spectrum.
Gels suitable for use in making a component of a device of the present invention
include but are not limited to agar gels, agarose gels, gelatins, low melting temperature
agarose gels, alginate gels, room-temperature Ca 2+ -induced alginate gels and
25 polysaccharide gels. Depending on the embodiment, a gel component has a water
content of greater than about 80% by weight, greater than about 92% by weight, greater
than about 95% by weight, greater than about 97% by weight and even greater than
about 98% by weight. In a preferred embodiment of the present invention, the gel
component includes an active entity. Suitable active entities include, but are not limited
30 to antibodies, antigens, biological materials, chemical materials, chromatogenic
compounds, drugs, enzymes, fluorescent probes, immunogenes, indicators, ligands,
nucleic acids, nutrients, peptides, physiological media, proteins, receptors, selective
toxins and toxins.
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In an embodiment of a device of the present invention, the cover is made of a gel
and the carrier is made of a non-gel material. Suitable non-gel materials include but are
not limited to elastically deformable materials, plastically deformable materials,
ceramics, epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
5 polydimethylsiloxane, polyethylenterephtalate glycol, polymers, polymethyl
methacrylate, paraffins, polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon
oxide, silicon rubbers and wax.
In an embodiment of a device of the present invention, the cover is made of a gel
and the carrier is made of a second gel. In such an embodiment, the gel from which the
10 cover is made and the second gel from which the carrier is made are substantially the
same or the two gels are substantially different.
In an embodiment of the present invention, the inside of the wells is configured
to delay adhesion of living cells thereto. In an embodiment of the present invention, the
inside of the well comprises a material that delays adhesion of living cells thereto, that
15 is the carrier is substantially fashioned from the adhesion-delaying material or the inside
of the wells is coated with the adhesion-delaying material. A suitable material to coat
the inside of the well or from which to make a carrier comprises polydimethylsiloxane,
is substantially polydimethylsiloxane or is substantially pure polydimethylsiloxane.
In a preferred embodiment of the present invention, the wells of the carrier are
20 juxtaposed. By juxtaposed is meant that in an area where wells are found, most of the
area is well area and little of the area is interwell .area. According to a feature of the
present invention, by juxtaposed is meant that the interwell area between two wells is
less than or equal to 0.35, 0.25, 0.15, 0.10 or even 0.06 of the sum of the areas of the
two wells. In certain embodiments of the present invention it is preferred that the
25 interwell area be substantially zero, that is that the rims of wells are substantially knife-
edged.
The dimensions of wells of a carrier of a chip-device of the present invention,
depending on the specific embodiment, are less than about 200 microns, less than about
100 microns, less than about 50 microns, less than about 25 microns or even less than
3 0 about 1 0 microns .
In a preferred embodiment of the present invention, the wells are configured to
hold no more than one living cell of a certain type. In another preferred embodiment,
the wells are configured to hold a predetermined number of living cells of a certain type.
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In an embodiment of the present invention, the wells are enclosures of
dimensions such that substantially at least one entire cell of a certain type (and
preferably no more than one such cell) is containable within such an enclosure, each
enclosure having an opening at the surface of the carrier, the opening defined by a first
5 cross section of a size allowing passage of a cell of the certain type. Depending on the
embodiment, the volume of such an enclosure is typically less than about 1 x 10" 11 liter,
less than about 1 x 10" 12 liter, less than about 1 x 1CT 13 liter, less than about 1 x 10' 14 liter
or even less than about 1 x 1CT 15 liter. Depending on the embodiment, the area of the
first cross section of such an enclosure is typically less than about 40000 micron 2 , less
10 than about 10000 micron 2 , less than about 2500 micron 2 , less than about 625 micron 2 or
even less than about 100 micron 2 .
In an embodiment of the present invention, the carrier further comprises
protuberances protruding from the surface between two adjacent wells, typically
between 1 micron and 10 microns high. In an embodiment of the present invention the
15 protuberances are sharp, for example, the area of the tip of the protuberances is less than
about 0,05 micron 2 . In an embodiment of the present invention the protuberances are
not sharp, for example, the area of the tip of the protuberances is between about 0.05
micron 2 and 28 micron 2 .
In an embodiment of the present invention, the carrier further comprises at least
20 one wall (preferably continuous) protruding from the surface, the at least one wall
circumscribing at least one area of the surface where the points of the top edge of the
wall define a plane.
In an embodiment of the present invention, the device further comprises a cover
slip configured to rest on the top edge of the least one wall so as to define at least one
25 closed volume including more than one well.
In an embodiment of the present invention, the device further comprises a cover
slip, and both the cover slip and the carrier are configured so as to allow the cover slip
to removeably rest above the surface of the carrier substantially in parallel to the surface
of the carrier. In an embodiment of the present invention, the carrier includes at least
30 one wall protruding from the surface, allowing the cover slip to rest thereupon
substantially in parallel to the surface. In an embodiment of the present invention, the
configuration of the carrier includes at least three protrusions protruding from the
surface, the protrusions having substantially the same height, allowing the cover slip to
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rest thereupon substantially in parallel to the surface. According to a feature of the
present invention, the cover slip and the carrier are configured so that there exist only a
limited number of correct cover slip positions wherein the cover slip is substantially
oriented in a specific position when resting above the surface.
5 According to the teachings of the present invention there is also provided a gel
carrier, the carrier, as described above, having a plurality of wells disposed on a surface
each well configured to hold at least one (and preferably no more than one) living cell
of a certain type. Different embodiments and features of a gel carrier of the present
invention are as described herein and as described hereinabove for a carrier of the chip
1 0 device of the present invention.
According to the teachings of the present invention there is also provided a
polydimethylsiloxane carrier, the carrier, as described above, having a plurality of wells
disposed on a surface each well configured to hold at least one (and preferably no more
than one) living cell of a certain type. Different embodiments and features of a
15 polydimethylsiloxane carrier of the present invention are as described herein and as
described hereinabove for a carrier of the chip device of the present invention.
According to the teachings of the present invention there is also provided a
carrier made of a first layer made of a first material resting on top of a second layer
made of a second material, the carrier having a plurality of wells disposed on an upper
20 surface of the first layer each well configured to hold at least one (and preferably no
more than one) living cell of a certain type, wherein the bottom of the plurality of wells
is the second layer. In a preferred embodiment, the second layer is made of a material
selected from the group consisting of ceramics, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol,
25 polymers, polymethyl methacrylate, polystyrene, polyurethanes, polyvinyl chloride,
silicon and silicon oxide, preferably glass. In a preferred embodiment, the first layer is a
fixed photoresist material. The different embodiments and features of such two-layered
carriers are as described herein and as described hereinabove for a carrier of the chip
device of the present invention described above.
30 According to the teachings of the present invention there is also provided a chip-
device for holding living cells, the device comprising a carrier having a plurality of
wells disposed on a surface, each well configured to hold at least one (and preferably no
more than one) living cell of a certain type, the carrier characterized in that bottoms of
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the wells are flat. In a preferred embodiment, the carrier comprises an additional feature
or features in addition to the wells, such as channels, fluid channels, fluid reservoirs,
microreactors, passages, plumbing routes, protruberances, transport channels and walls.
Preferably, at least some of the features are also flat-bottomed. Different embodiments
5 and features of such a flat-bottomed carrier are as described herein and as described
hereinabove for a carrier of the chip device of the present invention described above.
According to the teachings of the present invention there is also provided a chip-
device for holding living cells, the device comprising a carrier having a plurality of
wells disposed on a surface each well configured to hold at least one (and preferably no
10 more than one) living cell of a certain type, the device characterized in that the carrier is
made of a material having an index of refraction similar to that of water. In a preferred
embodiment of the present invention, the index of refraction of the carrier is less than
about 1.4, less than about 138, less than about 1.36, less than about 1.35, less than
about 1.34 or substantially equal to that of water. In an embodiment of the present
15 invention, such a carrier is made of a gel, preferably a transparent gel, preferably a
hydrogel. Suitable gels include agar gels, agarose gels, gelatins, low melting
temperature agarose gels, alginate gels, room-temperature Ca 2+ -induced alginate gels
and polysaccharide gels. Depending on the embodiment, a gel carrier has a water
content of greater than about 80% by weight, greater than about 92% by weight, greater
20 than about 95% by weight, greater than about 97% by weight and even greater than
about 98% by weight. Different embodiments and features of a earner of the present
invention having an index of refraction similar to that of water are as described herein
and as described hereinabove for a carrier of the chip device of the present invention.
According to the teachings of the present invention there is also provided a
25 device for holding living cells, the device comprising: (a) a well-bearing component
having a plurality of wells disposed on a surface each well configured to hold at least
one (and preferably no more than one) living cell of a certain type; and (b) a cover
covering the surface, the cover substantially made of a gel, preferably a transparent gel,
preferably a hydrogel. Suitable gels include agar gels, agarose gels, gelatins, low
30 melting temperature agarose gels, alginate gels, room-temperature Ca 2+ -induced
alginate gels and polysaccharide gels. Depending on the embodiment, a gel carrier has a
water content of greater than about 80% by weight, greater than about 92% by weight,
greater than about 95% by weight, greater than about 97% by weight and even greater
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than about 98% by weight. In a preferred embodiment of the gel-cover device of the
present invention, the gel includes an active entity. The dimensions of wells of a device
of the present invention having a gel cover, depending on the embodiment are less than
about 200 microns, less than about 100 microns, less than about 50 microns, less than
about 25 microns or even less than about 10 microns. Different embodiments and
features of a device of the present invention having a gel cover are as described herein
and as described hereinabove for the chip device of the present invention.
More generally, according to the teachings of the present invention there is also
provided a gel cover for wells of a well-bearing component of a well-bearing device (as
described herein and in the introduction) having a plurality of wells disposed on a
surface each well configured to hold at least one (and preferably no more than one)
living cell of a certain type.
According to the teachings of the present invention there is provided a method
of making a chip-device, or other devices and carriers of the present invention
comprising: (a) providing a template (such as a mold or stamp) having a negative of
features of the surface of the carrier; (b) contacting the template with a precursor
material so as to create the features in the precursor material; and (c) fixing the features
in the precursor material so as to fashion the carrier.
Depending on the embodiment and the nature of the precursor material, fixing
includes such methods a heating the precursor material, cooling the precursor material,
polymerizing the precursor material, cross-linking the precursor material, curing the
precursor material, irradiating the precursor material, illuminating the precursor
material, gelling the precursor material, exposing the precursor material to a fixative and
waiting a period of time.
The template is preferably made of a material that is rigid compared to the
precursor material. Suitable materials include but are not limited to elastically
deformable materials, plastically deformable materials, ceramics, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate, paraffins,
polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon oxide, silicon rubbers
and wax.
Features created in the precursor material include such features as wells,
channels, coupling elements, drains, fluid channels, fluid reservoirs, input ports, light
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sources, magnetizable elements, membranes, microreactors, microvalves, passages,
optical components, optical fibers, optical filters, output ports, plumbing routes,
protruberances, pumps, transport channels, valves, walls and fiducial points.
In an embodiment of the present invention, one of the features is a fiducial point
5 and prior to fixing the features, a marking material (e.g., metals, fluorescent materials
and visible materials) is added to the incipient fiducial point. A preferred method of
adding a marking material is by applying the marking material onto the respective
negative of the fiducial point before contacting the template with the precursor material.
In an embodiment of the present invention, subsequent to fixing the features in
10 the precursor material the template is separated from the carrier and additional device
components are attached to the carrier. Examples of such additional device components
include but are not limited to cover slips, piping, tubing, pumps, fluid supplies and
observation components. Attaching can include the use of methods employing
adhesives or surface treatments such as plasma treatments.
15 In an embodiment of the present invention the precursor material is a plastically
deformable material (vide infra) such as a wax, a paraffin, plastic or polymer, and fixing
the features simply includes separating the template from the precursor material.
In an embodiment of the present invention the precursor material is an elastically
deformable material (vide infra) such as a gellable fluid, a polymerizable material, a
20 powder, a fluid or a thermoplastic material.
In an embodiment of the present invention, the elastic precursor material is a
themioplastic material at plastic temperature and fixing the features includes cooling the
thermoplastic material.
In an embodiment of the present invention, the elastic precursor material is a
25 polymerizable material and fixing the features includes polymerizing the polymerizable
material. Suitable polymerizable materials include but are not limited to monomer
solutions, crosslinkable polymers, vulcaoizable polymers, polymerizable fluid and
thermosetting resins.
In a preferred embodiment, the polymerizable material is a polydimethylsiloxane
30 precursor mixture and fixing the features includes polymerizing the
polydimethylsiloxane precursor mixture so as to produce polydimethylsiloxane. In
another preferred embodiment, the polymerizable material includes urethane and fixing
the features includes polymerizing the urethane to produce polyurethane.
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In an embodiment of the present invention, the elastic precursor material is a
gellable fluid and fixing the features includes gelling the gellable fluid. Depending on
the nature of the gellable fluid used, preferred methods of gelling the gellable fluid
include of heating the gellable fluid, cooling the gellable fluid, irradiating the gellable
5 fluid, illuminating the gellable fluid, contacting the gellable fluid with a gelling reagent
and waiting a period of time for the gellable fluid to gel. Suitable gellable fluids include
but are not limited to agars, agaroses, gelatins, low melting temperature agaroses,
alginates, room-temperature Ca 2+ -inducable alginates and polysaccharides. A preferred
gellable fluid is an alginate solution where gelling the gellable fluid includes contacting
10 the gellable fluid with a gelling reagent, such as a gelling reagent including Ca 2+ ions.
An additional preferred gellable fluid is a low melting temperature agarose solution and
gelling the gellable fluid includes cooling the gellable fluid.
According to the teachings of the present invention there is also provided a
method of making a chip-device, or other devices and carriers of the present invention
15 comprising: (a) providing a carrier having a plurality of wells disposed on a surface,
each well configured to hold at least one (and preferably no more than one) living cell
of a certain type; and (b) coating the inside of the wells with a layer of a material
configured to influence proliferation of living cells held in the wells.
In an embodiment of the present invention, coating the inside of the wells
20 comprises (i) applying a precursor fluid to the inside of the wells; and (ii) solidifying the
precursor fluid so as to form the proliferation-influencing layer, Suitable methods of
solidifying include but are not limited to heating the precursor fluid, cooling the
precursor fluid, polymerizing the precursor fluid, cross-linking the precursor fluid,
curing the precursor fluid, irradiating the precursor fluid, illuminating the precursor
25 fluid, gelling the precursor fluid, exposing the precursor fluid to a fixative and waiting a
period of time.
In another embodiment of the present invention, coating the inside of the wells
comprises (i) depositing a vapor of the material onto the surface thereby forming the
proliferation-influencing layer.
30 In another embodiment of the present invention, coating the inside of the wells
comprises (i) depositing a vapor of a precursor material onto the surface; and (ii)
solidifying the precursor material thereby forming the proliferation-influencing layer.
Suitable methods of solidifying include but are not limited to heating the precursor
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fluid, cooling the precursor fluid, polymerizing the precursor fluid, cross-linking the
precursor fluid, curing the precursor fluid, irradiating the precursor fluid, illuminating
the precursor fluid, gelling the precursor fluid, exposing the precursor fluid to a fixative
and waiting a period of time. In a preferred embodiment, the vapor of precursor material
is a vapor of para-xylylene molecules or derivatives thereof and the layer comprises the
polymerized para-xylylene molecules (or derivatives thereof). By para-xylylene
derivatives is meant a a molecule that is substantially a para-xylylene molecules having
any additional substituent on either or both the aromatic rings
According to a feature of the present invention, the surface of the carrier is made
of a material including but not limited to elastically defonnable materials, plastically
deformable materials, ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon
oxide and silicon rubbers.
The devices and carriers of the present invention allow performance of a variety
of heretofore difficult or impossible to perform experiments.
According to the teachings of the present invention, there is provided a method
of manipulating cells comprising: (a) providing a plurality of wells of a well-bearing
component, each well configured to hold at least one (and preferably no more than one)
living cell of a certain type; (b) holding a plurality of living cells in a plurality of the
wells; (c) placing a gellable fluid in proximity with the surface so as to fill the plurality
of wells; and (d) gelling the gellable fluid so as to form a gel cover. The method of
manipulating cells is generally and is applicable to well-bearing components such as
described hereinabove (including in the introduction), especially carriers, especially
carriers of the present invention. For greatest utility it is preferred that each of the wells
be individually addressable, that the wells be juxtaposed, and that the bottoms of the
wells be coplanar. Gelling the gellable fluid so as to form the gel cover is performed
using a number of methods including heating the gellable fluid, cooling the gellable
fluid, irradiating the gellable fluid, illuminating the gellable fluid, contacting the
gellable fluid with a gelling reagent and waiting a period of time for the gellable fluid to
geL
According to a feature of the present invention, placing the gellable fluid
comprises, i) placing a plurality of cells in a gellable fluid in the proximity of the wells;
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and ii) causing the cells to settle into the wells so as to be held in respective wells.
Generally, causing the cells to settle into the wells includes applying a force to the cells,
typical forces including gravitation, centrifugal forces, forces resulting from the impact
of photons on the cells (e.g., laser tweezers, application of a non-focussed laser (see, for
example, P.A.L.M, Microlaser Technologies AG, Bemried, Germany)), or forces
resulting from a pressure wave (such as produced by an ultrasonic transponder).
In an embodiment of the present invention, the well-bearing component is a
carrier made of a gel. In an embodiment of the present invention, the gel formed
subsequent to gelling is transparent. In an embodiment of the present invention, the gel
formed is a hydrogel. Suitable gellable fluids for making a gel cover of the present
invention include but are not limited to agars, agaroses, gelatins, low melting
temperature agaroses, alginates, room-temperature Ca 24 -inducable alginates and
polysaccharides. A preferred gellable fluid is an alginate solution where gelling the
gellable fluid includes contacting the gellable fluid with a gelling reagent, such as a
gelling reagent including Ca 2+ ions. An additional preferred gellable fluid is a low
melting temperature agarose solution and gelling the gellable fluid includes cooling the
gellable fluid.
In a preferred embodiment, prior to gelling, substantially every one of the wells
holds no more than one cell.
In an embodiment of the method of the present invention, the inside of the wells
is a proliferation-delaying, such as a gel or a hydrogel In an embodiment of the method
of the present invention, the inside of the wells is an adhesion-delaying surface. Such a
surface includes polydimethylsiloxane, is substantially polydimethylsiloxane or is
substantially pure polydimethylsiloxane.
Li an embodiment of the present invention, subsequent to gelling of the gel
cover, at least one held cell is isolated by excising the at least one cell from the well-
bearing component.
In one embodiment of the present invention, the gellable fluid includes an active
entity. Suitable active entities include, but are not limited to antibodies, antigens,
biological materials, chemical materials, chromatogenic compounds, drugs, enzymes,
fluorescent probes, immunogenes, indicators, ligands, nucleic acids, nutrients, peptides,
physiological media, proteins, receptors, selective toxins and toxins.
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In one embodiment of the present invention, subsequent to gelling the gellable
fluid, an active entity containing fluid is contacted with the produced gel cover. If the
well-bearing component (such as a carrier) is also a gel, then an active entity containing
fluid is also or exclusively contacted with the gel well-bearing component. Suitable
5 active entities include, but are not limited to antibodies, antigens, biological materials,
chemical materials, chromatogenic compounds, drugs, enzymes, fluorescent probes,
immunogenes, indicators, ligands, nucleic acids, nutrients, peptides, physiological
media, proteins, receptors, selective toxins and toxins. Subsequent to the contact of the
active-entity, a period of time is waited so as to allow the active entity to diffuse
10 through the gel cover (or gel well-bearing component).
In one embodiment of the present invention, subsequent to gelling the gellable
fluid, the cells are allowed to proliferate into or through the produced gel cover. If the
well-bearing component (such as a carrier) is also a gel, then the cells are allowed to
proliferate into or through the gel well-bearing component. Although gels have cell-
15 proliferation delaying properties, after some time cells do proliferate into and through
gels.
According to the teachings of the present invention there is also provided a
method of growing cells comprising: (a) providing a well-bearing device; (b) holding at
least one (and preferably no more than one) living cell of a certain type in a well of the
20 well-bearing device (preferably a chip device, especially a chip device of the present
invention having a carrier with changeable well-sizes described hereinabove); and (c)
increasing the size of the well so as to provide an increased space for proliferation of the
cell.
In an embodiment of the present invention, prior to increasing the size of the
25 wells, a plurality of cells is held in a plurality of wells of the well-bearing device; and
subsequently the size of the wells increased so as to provide an increased space for
proliferation of living cells. Preferably each of the wells holds no more than one cell. In
an embodiment of the present invention, discarding includes physically moving at least
one of the non-selected cells, for example using optical-tweezers. In an embodiment of
30 the present invention, discarding includes damaging at least one of the non-selected
cells, for example by irradiating with a laser.
According to the teachings of the present invention there is provided a method
of collecting cells (preferably living cells) from a biological sample (such as a tumor,
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organ, flesh or tissue) comprising: (a) providing a well-bearing device, the well-bearing
device having: (i) a plurality of wells disposed on a surface, each well configured to
hold at least one cell of a certain type (and preferably no more than one); and (ii) a
plurality of protuberances (sharp or not sharp) protruding from the surface (b)
contacting the biological sample with the surface so as to remove cells from the
biological sample. Preferably the well-bearing device is a chip-device, especially a chip
device of the present invention. Preferably the surface is a surface of a carrier. Typical
protuberances are made of a material such as plastic, polymer, metal or glass. In an
embodiment of the present invention, the protruberances protrude from areas between
the wells. Preferably, prior to the contacting of the biological sample, substantially all of
the wells are filled with a fluid such as water, physiological fluid and physiological
media.
Preferably, subsequent to the contacting of the biological sample, a cover is
placed on top of the surface.
In an embodiment of the present invention, to increase the yield of cells
collected, a trauma is applied to the biological sample. Suitable trauma include
crushing, cutting, macerating, mashing, slicing and squeezing the biological sample. In
an embodiment of the present invention, during the contacting, a flow of fluid is
provided along the biological sample so as to increase the yield of cells collected.
Unless otherwise defined, all technical and scientific terms used herein have the
same me aning as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the present invention, suitable
methods and materials are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the
accompanying drawings. With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present invention only, and
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are presented in the cause of providing what is believed to be the most useful and
readily understood description of the principles and conceptual aspects of the invention.
In this regard, no attempt is made to show structural details of the invention in more
detail than is necessary for a fundamental understanding of the invention, the
5 description taken with the drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
In the drawings:
FIG. 1 (prior art) depicts a cell-chip device of PCT patent application
IL01/000992 including a transparent carrier;
10 FIG. 2 (prior art) is a reproduction of a photograph of a cell-chip device of PCT
patent application IL01/000992;
FIG. 3 (prior art) is a reproduction of a photograph of a cell-populated well
matrix of a carrier of a cell-chip device of PCT patent application IL01/000992;
FIG. 4 is a reproduction of a scanning electron micrograph of the domes on a
15 nickel stamp used for the production of a carrier of the present invention;
FIG." 5 is a reproduction of a scanning electron micrograph of a well-matrix of a
polydbnethylsiloxane carrier manufactured according to the method of the present
invention using the nickel stamp of Figure 4;
FIGS. 6A-6C schematically depict a method of the present invention where a gel
20 carrier is manufactured according to the method of the present invention and where a
gel cover is manufactured according to the method of the present invention;
FIGS. 7A-7C schematically depict the use of an elastically deformable carrier of
the present invention;
FIGS. 8A-8D schematically depict the use of an elastically deformable carrier of
25 the present invention;
FIG. 9A is a reproduction of a scanning electron micrograph of a well-matrix of
a carrier of the present invention having sharp protuberances protruding from the
surface of the carrier;
FIG. 9B is a reproduction of a scanning electron micrograph of a well-matrix of
30 a carrier of the present invention having non-sharp protuberances protruding from the
surface of the carrier; and
FIGS. 10A-10B schematically depict the method of collecting cells of the
present invention.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention is of a device that is substantially a well-bearing
component for use in isolating cells, configured to influence the proliferation of living
cells held in the wells. The present invention is also of a method of producing a device
5 of the present invention. The present invention is also of a method of manipulating cells
by holding the cells in wells of a well-bearing component under a gel cover and then
separating selected cells from other cells, transporting the well-bearing device, adding
active entities through the gel cover and allowing the cells to proliferate into the gel.
The present invention is also of a method of allowing cell proliferation by holding the
10 cells in wells of a well-bearing component and then increasing the size of the wells. The
present invention is also of a method for collecting cells from a biological sample.
The principles and uses of the teachings of the present invention may be better
understood with reference to the accompanying description, figures and examples. In
the figures, like reference numerals refer to like parts throughout.
15 Before explaining at least one embodiment of the invention in detail, it is to be
understood that the invention is not limited in its application to the details set forth
herein. The invention can be implemented with other embodiments and can be practiced
or carried out in various ways. It is also understood that the phraseology and
terminology employed herein is for descriptive purpose and should not be regarded as
20 limiting.
Unless otherwise defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the present invention, suitable
25 methods and materials are described below. All publications, patent applications,
patents and other references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the patent specification, including definitions, will control.
In addition, the materials, methods, and examples are illustrative only and not intended
to be limiting.
30 The terms "comprising" and "including" or grammatical variants thereof when
used herein are to be taken as specifying the stated features, integers, steps or
components but do not preclude the addition of one or more additional features,
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integers, steps, components or groups thereof. This term encompasses the terms
"consisting of and "consisting essentially of \
The phrase "consisting essentially of means that the composition may include
additional ingredients, but only if the additional ingredients do not materially alter the
basic and novel characteristics of the claimed compositions or methods.
The term "method" refers to manners, means, techniques and procedures for
accomplishing a given task including, but not limited to, those manners, means,
techniques and procedures either known to, or readily developed from known manners,
means, techniques and procedures by practitioners of the chemical, pharmacological,
biological, biochemical and medical arts.
Hereinfurther, the term "active entity" is understood to include chemical,
biological or pharmaceutical entities including any natural or synthetic chemical or
biological substance that influences a cell with which the entity is in contact. Typical
active entities include but are not limited to active pharmaceutical ingredients,
antibodies, antigens, biological materials, chemical materials, chromatogenic
compounds, drugs, enzymes, fluorescent probes, immunogenes, indicators, ligands,
nucleic acids, nutrients, peptides, physiological media, proteins, receptors, selective
toxins and toxins.
Implementation of the methods of the present invention involves performing or
completing selected tasks or steps manually, automatically, or a combination thereof
Device of the present invention
A first device of the present invention is a chip-device for holding living cells
resembling the chip device described in PCT patent application IL0 1/000992, the device
including, together with other components, a carrier having a plurality of wells disposed
on a surface of the carrier, each well configured to hold at least one (and preferably no
more than one) living cell of a certain type, the device characterized in that the wells are
configured to influence the proliferation of living cells held in the wells. Whereas prior
art carriers may have had some characteristics that incidentally influence the
proliferation of cells, in the present invention the influence is predetermined.
Specifically desired features include that the inside of the wells is configured to delay
(or inhibit) cell proliferation, to delay (or inhibit) cell adhesion, that the wells are
configured so as to allow cell-proliferation inside and through at least one component of
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the chip-device, that the carrier has an index of refraction similar to that of water or that
the size of the wells is changeable. A preferred means for implementing the teachings of
the present invention is by coating the inside of the wells (with which held cells are
potentially in contact) or by fashioning the carrier substantially in entirety from
5 materials such as gels (especially hydrogels), polydimethylsiloxane or an elastomer
such as silicon rubber.
As discussed hereinabove, a problem in the art is that there exists no simple way
to allow cells that have been isolated and selected, for example according to the
teachings of PCT patent application IL0 1/00992, to proliferate freely. On the one hand,
10 for efficient selection and isolation, wells are necessarily small, too small to allow cells
to proliferate therein. It is extremely difficult and inefficient to extract a single selected
cell from a prior art well-bearing device and to place the cell in a sufficiently large
location to allow proliferation. When, in accordance with the teachings of PCT patent
application IL0 1/00992, a plurality of different cells are selected and isolated in one
15 well-bearing device it is considerably more difficult to relocate each one of the plurality
of cells to a remote location for proliferation.
Therefore, in one embodiment of the present invention, the size of the wells of a
device of the present invention is changeable. The changeability of the wells is achieved
by configuring a carrier of the present to be deformable in at least one dimension
20 (length, breadth, depth, any two or all three) and that upon deformation, the size of at
least one of the wells on the carrier is changed. Generally the deformation is by
stretching and generally the change of size of a well is an increase of size of the well.
In one embodiment of the present invention the carrier is elastically deformable,
for example, the carrier is made substantially of an elastically deformable material
25 including but not limited to elastomers, rubber, silicon rubbers or other materials, for
example elastic materials listed in U.S. Patent 6,740,727, U.S. Patent 6,682,792 and
U.S. Patent 6,673,857. By elastically deformable material is meant a material that is
capable of recovering shape after deformation. For example, a suitable elastomer that is
commercially available is Silastic® LSR 9280-30 (Dow Corning Corporation, Midland,
30 MI, USA). Such elastically deformable carriers may be generally placed in a deforming
device and by the application of tension stretched to a desired extent. The elastically
deformable carrier remains in a deforming device and the tension maintained for as long
as the changed size is desired.
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In another embodiment of the present invention, the carrier is plastically
deformable, that is the carrier is made of a plastically deformable material. By
plastically deformable material is meant a material where the original topology is
substantially maintained during deformation but does not recover shape after
deformation. Such plastically deformable carriers are generally placed in a deforming
device and by the application of tension stretched to a desired extent while topology
such as surface features is substantially maintained throughout the deformation process.
Tension can be released as the carrier does not recover the former shape. Plastically
deformable materials useful in implementing a carrier of the present invention include
but are not limited to hydrocarbon waxes (such as PARAFILM®, Pechiney Plastic
Packaging, Inc., Neenah, WI, USA), crystalline wax, polypropylene, isotactic
polypropylene homopolymer, syndiotactic polypropylene homopolymer, metallocene
catalyzed isotactic polypropylene homopolymer, metallocene catalyzed syndiotactic
polypropylene homopolymer, ethylene-propylene random copolymer, butene-propylene
random copolymer, ethylene-propylene-butene-1 terpolymer, low density polyethylene,
linear low density polyethylene, very low density polyethylene, metallocene catalyzed
polyethylene, metallocene catalyzed polyethylene copolymers, ethylene-methacrylate
copolymers, ethylene-vinyl acetate copolymers, ionomer resins, an ethylene-propylene
random copolymer, ethylene-butene-1 copolymer, ethylene-propylene-butene-1
terpolymer, propylene-butene copolymer, low density polyethylene, linear low density
polyethylene, very low density polyethylene, metallocene catalysed polyethylene
plastomer, metallocene catalyzed polyethylene, metallocene catalyzed polyethylene
copolymers, ethylene-methacrylate copolymer, ethylene vinyl acetate copolymer,
ionomer resin and combinations thereof.
The use of device of the present invention having changeable size wells is
discussed in detail hereinbelow.
A further material that is used in producing a component of a device of the
present invention for implementing the teachings of the present invention is a gel,
especially a hydrogel. Suitable gels include but are not limited to hydrogels, agar,
gelatin, agarose gels, low melting temperature agarose gels, alginate gels, room-
temperature Ca 2+ -induced alginate gels and polysaccharide gels. The components of the
device of the present invention that are advantageously made of gel are either a carrier,
a cover for the carrier or both. Embodiments of the device of the present invention
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include embodiments where both the carrier and the cover aremade of gel. In such
cases, the gel from which the carrier is made and the gel from which the cover are made
may be substantially identical or may have different compositions. Suitable gels for the
carrier, the cover or both include but are not limited to gels, hydrogels, agar gels,
agarose gels, gelatins, low melting temperature agarose gels, alginate gels, room-
temperature Ca 2+ -induced alginate gels and polysaccharide gels. Embodiments of the
device of the present invention include embodiments where both the cover is made of a
gel (as described above) and the carrier is made of another material. Suitable materials
from which carriers are made include but are not limited to elastically deformable
materials, plastically deformable materials, ceramics, epoxies, glasses, glass-ceramics,
metals, plastics, polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol,
polymers, polymethyl methacrylate, paraffins, polystyrene, polyurethanes, polyvinyl
chloride, silicon, silicon oxide, silicon rubbers and waxes.
The advantages of making components of a device of the present invention from
gels are manifold. Certain gels may have cell-proliferation delaying properties: a cell
that is encased in such a gel does not significantly proliferate for a period of two to three
days. However, once a cell begins to proliferate, proliferation occurs into and through
the gel matrix with little interference. Thus a cell encased in a gel can proliferate
without the overcrowding problems discussed hereinabove.
As discussed hereinabove, one problem of transparent devices known in the art
is that the index of refraction of the materials from which the wells are made is
significantly greater than that of water. For example glass has an index of refraction of
1.5 whereas water or physiological media have an index or refraction of only about
1.33. Considering the curvature and the dimensions of elements and features of well-
bearing components of devices known in the art, the difference in index of refraction is
significant enough to cause scattering, reflection and diffraction of light, interfering
with optical study of held cells, for example, during morphological studies using a
microscope. Thus, more generally, in an embodiment of the present invention the well-
bearing component is substantially made of a material that has an index of refraction
similar to the index of refraction of water or physiological medium. By an index of
refraction similar to the index of refraction of water is meant herein an index of
refraction of less than about 1.4, preferably less than about 1.38, more preferably less
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than about 136, more preferably less than about 1.35 and even more preferably less
than about 134, or substantially identical to that of water.
Therefore, in accordance with the teachings of the present invention, optical
study of a cell through a component made of a transparent gel is exceptionally effective
5 as gels, especially hydrogels, have an index of refraction substantially identical to the
index of refraction of water and physiological media. As a result, when observing cells
held in a gel, the gel components are substantially transparent and only the cells are
seen. Gels that are substantially transparent to visible light, ultraviolet light and infrared
radiation are known.
10 Although any type of gel can be used for implementing the teachings of the
present invention, hydrogels are preferred. Hydrogels are gels having a high percentage
of water. Typical hydrogels useful for implementing the teachings of the present have a
water content of greater than 80% by weight, greater than 92% by weight, greater than
95% by weight, greater than 97% by weight and even greater than 98% by weight.
15 As will be discussed in detail hereinfurther, in general a gel cover of the present
invention is advantageously produced by placing a gellable fluid over the top surface of
a well-bearing component and subsequently gelled, sandwiching the cells between the
wells and the gel cover. It is therefore preferred that a gel used be produced from a
gellable fluid that is fluid and gels under conditions that are not damaging to a cell. Two
20 exceptionally preferred types of hydrogels are alginates and low melting temperature
agaroses.
Alginates are biologically compatible polysaccharide proteins that are fluid at
low calcium ion concentrations (e.g., [Ca 2+ ] < 1 jxM) but gel upon exposure to higher
concentrations of calcium ions {e.g., [Ca 2+ ] = 20 mM). An exceptionally suitable
25 alginate for implementing the teachings of the present invention is sodium alginate and
may be purchased, for example, from Pronova Biopolymers (Drammen, Norway) as
Protanal LF120 1% in water or Protanal LF200 1% in water.
Low melting temperature agaroses are biologically compatible gels that before
gelling are fluid at temperatures that do not harm living cells (e.g. , 20°C), gel at low
30 temperatures that do not harm living cells (e.g., 4°C) and remain stable until well-above
temperatures used for studying living cells (40°C). An exceptionally suitable agarose for
implementing the teachings of the present invention that may be purchased, for
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example, from Cambrex Bio Science Rockland Inc. (Rockland, ME, USA) is HGS-
LMP Agarose (catalogue nr. 50221).
An additional advantage of a gel component of a device of the present invention,
such as a carrier or a cover, is that it is possible to include an active entity, such as those
discussed hereinabove, in the gel.
In a further embodiment, the inside of the wells (the physical surface of the well)
of the carrier of a device of the present invention are configured to delay cell adhesion
of living cells thereto. In one embodiment, the carrier is fashioned substantially from an
adhesion-delaying material. In another embodiment, the carrier is fashioned from some
material and the inside surface of the wells with which held cells potentially make
contact is coated with an adhesion-delaying material.
In some embodiments of the present invention, a preferred material with which
to coat a carrier or from which to make a carrier includes polydimethylsiloxane. When a
coating material or the material from which the carrier is made includes
polydimethylsiloxane, the polydimethylsiloxane is optionally one of the adhesion-
delaying or inhibiting materials, is substantially the adhesion-delaying or inhibiting
material or the material is substantially pure polydimethylsiloxane.
Substantially pure polydimethylsiloxane is a cross-linked polymer characterized
by good optical transparency, low fluoresence, thermal and environmental stability and
is inert to most laboratory reagents. Polydimethylsiloxane is not-cytotoxic. Importantly,
polydimethylsiloxane has been found to delay cell adherence, thus delaying cell
proliferation, see below. Suitable polydimethylsiloxane resins are commercially
available and can be purchased, amongst others, under the trade names RTV615 PDMS
(GE Silicones, Wilton, CT, USA) and Sylgard 184 PDMS (Dow Corning Corporation,
Midland, MI, USA).
A device of the present invention advantageously incorporates and includes
many of the innovative features disclosed in PCT patent application IL0 1/000992.
Preferred such features are discussed hereinfurther.
In embodiments of the device of the present invention it is preferred that each
well be individually addressable.
In embodiments of the device of the present invention, the wells are defined by
an intersection of at least two channels on the surface of the carrier. Preferably, the at
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least two intersecting channels are transport channels configured to transport fluids from
one location of the carrier to another location of the carrier.
To increase loading of cells per unit area, it is preferred that the wells of a carrier
of a device of the present invention be round or hexagonal and be hexagonally packed.
Other preferred well shapes include square, triangular and rectangular wells.
In embodiments of the device of the present invention, the wells are juxtaposed.
By juxtaposed is meant that in an area where wells are found, most of the area is well
area and little of the area is interwell area. As disclosed in PCT patent application
ELG1/000992, when hexagonal wells are hexagonally packed, then a carrier can be
fashioned so that the total interwell area between any cluster of seven wells is less than
or equal to about 0.35, 0.25, 0.15, 0.10 or 0.06 the sum of the areas of the seven wells.
This is more generally expressed herein in that the interwell area between two wells is
less than or equal to 0.35, 0.25, 0.15, 0.10 or 0.06 of the sum of the areas of the two
wells. In certain embodiments of the present invention it is preferred that the interwell
area be substantially zero, that is that the rims of wells are substantially knife-edged.
In a typical example of a 2mm x 2mm matrix of hexagonal knife-edged wells of
the present invention, where each well is about 10 microns wide, there are 61600 wells,
a well density of about 1.5 x \0 6 wells cm" 1 .
The wells of a device of the present invention are generally of any size so as to
hold at least one cell of a certain type. As the teachings of the present invention are
directed to cellular biology, it is generally preferred that the wells be small so as to
avoid having a large number of cells held in any one well. Thus, generally, the
dimensions of the wells are generally less than about 200, 100, 50, 25 or even 10
microns. By dimensions is meant the usual meaning of the word and is dependent on the
shape of the well. For example, for hexagonal or circular wells, the term dimension
refers to diameter. For square or triangular wells is meant the longest dimension of the
square or triangle, respectively. The exact size of wells of any given device is
determined by the type of cells or alternately or additionally by the amount of cells to be
studied using the device. Since different types of cells have different sizes, generally a
device of the present invention will have wells of a. size to accommodate one or more
cells of the type to be studied. Most preferred is that a well be of a size so as to hold no
more than one cell of the type to be studied at any one time. In other embodiments, a
well size is determined by the size of a predetermined number of a certain type of cells.
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In embodiments of the device of the present invention, the bottoms of the wells
of a carrier are preferably coplanar. This is exceptionally true when the wells are
configured to hold only one cell of a certain type: coplanarity allows for optical
observation of many cells (whether by scanning or simultaneously using a wide-angle
5 observation component) without the need for time consuming and technically difficult
to implement refocusing.
In some embodiments of the present invention, wells are dimples or depressions
on the surface of the carrier. In other embodiments, the wells are substantially
enclosures of dimensions such that substantially an entire cell of a certain type is
10 containable within the enclosure, each enclosure having an opening at the surface, the
opening defined by a first cross section of a size allowing passage of a cell of the certain
type. The volume of such enclosure wells is typically less than 1 x 10" 11 liter
(corresponding to the volume of a 200 micron cube), less than 1 x 10~ 12 liter
(corresponding to the volume of a 100 micron cube), less than 1 x 10" 13 liter
15 (corresponding to the volume of a 50 micron cube), less than 1 x 10" 14 liter
(corresponding to the volume of a 25 micron cube) and even less than 1 x 10" 15 liter
(corresponding to the volume of a 10 micron cube). In a preferred embodiment of the
present invention, the dimensions of an enclosure are such as to contain no more than
one cell of a certain size at any one time. The area of the first cross section,
20 corresponding to the size of the opening of a respective enclosure is typically less than
about 40000 micron 2 (corresponding to the area of a 200 micron square), 10000 micron 2
(corresponding to the area of a 100 micron square), 2500 micron 2 (corresponding to the
area of a 50 micron square), 625 micron 2 (corresponding to the area of a 25 micron
square) or even less than about 100 micron 2 (corresponding to the area of a 10 micron
25 square).
In some embodiments, the surface of the carrier is substantially transparent so as
to allow observation of cells while the lower surface is substantially not transparent. In
some embodiments, the lower surface of the carrier is substantially transparent while the
surface is substantially not transparent. In some embodiments, both the surface and the
30 lower surface of the carrier are substantially transparent. In some embodiments, both the
surface and the lower surface of the carrier are substantially not transparent. By
transparent is especially meant transparent to one or more frequencies of
electromagnetic radiation in the visible, ultraviolet or infrared spectra.
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In some embodiments of the present invention, there is at least one fluid
transport channel on the surface of the carrier, the fluid transport channel configured to
transport fluids from one location of the carrier to another location of the carrier. In
some embodiments of the present invention, there are fluid transport channels on the
surface configured to transport fluids from one well to another well. In some
embodiments of the present invention, a fluid transport channel separates one group of
wells from another group of wells.
In some embodiments of the present invention there are channels for
transporting fluids from the surface to the lower surface through the carrier. In some
instances, the channels are pores in the wells (especially at the bottom of the wells), the
pores being of a size so as to prevent passage of cell of the type to be studied
therethrough.
In some embodiments of the present invention a device of the present invention
has a cover slip as a component. For use, a cover slip is positioned above and generally
substantially parallel with the surface of a respective carrier. The cover slip provides a
closed volume and seals fluid transport channels and such-like features of the carrier so
that fluids can be directed to flow as desired. In some embodiments a cover slip is
attached to a respective carrier. Attaching is performed, for example, using an adhesive
or a surface treatment such as plasma treatment. There exist many suitable adhesives,
including but not limited to light curable adhesives, for example light curing adhesive
3051 or 3341 manufactured by Henkel Loctite Deutschland GmbH, Munchen,
Germany.
In a preferred embodiment, the device, the cover slip and the carrier are
configured so as to allow the cover slip to removeably rest above the surface of the
carrier substantially in parallel to the surface of the carrier. In some embodiments, the
cover slip and the carrier are configured so that there exist only a limited number (e.g.,
six, four, three, two or even one) of "correct" cover slip positions where the cover slip is
substantially oriented in a specific position when resting above the surface. Such a
limited number depends on the shape and design of features of the carrier and
specifically the shape and arrangement of the wells thereof.
There exist many reasons to design a cover slip having only a limited number of
orientations. In a preferred embodiment, a cover slip is provided with one or more cover
slip microelectrode, as detailed in PCT patent application IL0 1/000992. A cover slip
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microelectrode is generally configured to be positioned substantially above and
associated with a specific well and, when activated, apply a repulsive force so as to push
cells downwards into the associated well or apply an attractive force so as to extract
cells out of the associated well. Clearly, for a cover slip microelectrode to be properly
positioned and properly addressable, the cover slip preferably has only a limited number
of correct positions.
In some embodiments of the present invention, a carrier is provided with a wall
or walls, similar to walls detailed in PCT patent application IL01/000992. The details of
shape and geometry of such a wall is dependent on the purpose such a wall serves. The
wall may be configured to encircle all of the wells or to isolate groups of wells. Such a
wall can be continuous or not. The top of the wall can define a plane. In some
embodiments, such a wall acts as a mold to assist in making a gel cover. The wall
surrounds the wells. When a gel cover is made by pouring a gellable fluid onto the
surface of the carrier (vide infra), the wall holds the gellable fluid in place until the
gellable fluid gels. In other embodiments of the present invention, such a wall helps
support a cover slip in the proper position, orientation and height above the carrier. In
other embodiments of the present invention, such a wall defines, together with a cover
slip, a volume containing one or more wells.
In some embodiments of the present invention a carrier is provided with
protuberances protruding from the surface, generally between two adjacent wells. The
details of shape and geometry of such protuberances is dependent on the purpose such
protuberances serve.
In some embodiments of the present invention, protuberances help support a
cover slip in the proper position, orientation and height above the carrier.
In some embodiments of the present invention it is desired that when the cover
slip is in place, fluids flow freely in the volume between the carrier and the cover slip,
but cells are prevented from doing so. In such an embodiment it is often advantageous
to provide protuberances between wells so that the size of the passage defined by the
cover slip, the protuberances and the carrier between wells is such that a cell cannot
pass therethrough acting, in fact, as a porous barrier to cell movement or fence.
In other embodiments, protuberances are used to implement the method of
collecting cells of the present invention (vide infra). In brief, a biological sample is
placed directly on a carrier provided with protuberances following the removal of a
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cover slip, if present. The protuberances assist the release of living cells from the
biological sample. Cells released from the sample settle directly into wells of the carrier.
Protuberances used for implementing the method of cell collection of the present
invention are of any height that is convenient for production. Typical protuberances
5 used for implementing the method of cell collection of the present invention are
between about 1 micron high and about 20 microns high. Such protuberances can be
sharp or not sharp. The term not sharp is a relative term, and depends on the dimensions
of the cells to be harvested from the biological sample. It has been found that
sufficiently not sharp so as not to pierce a cell under the conditions used but still
10 effectively assist in removal of the cell from the biological matrix, a not sharp
protuberance is generally of a width between about 5% and about 30% of the cell
diameter, or preferably between about 10% and about 20% of the cell diameter.
A device of the present invention is advantageously provided with a flow
generator configured to generate a flow of fluid substantially parallel to the surface of
15 the carrier. As discussed in PCT patent application IL0 1/000992, a parallel fluid flow is
useful for washing away cells that are not held in wells.
A device of the present invention may be used in conjunction with or is
advantageously provided with optical tweezers and similar devices, configured to mo ve
cells found in the proximity of a carrier of the present invention. Optical tweezers can
20 be used to push cells into wells or to extract cells therefrom.
The innovative use of gel as a cover as disclosed herein is not limited to a device
of the present invention or to use with a carrier of the present invention. Rather, the use
of a gel is useful for any device for holding living cells where the device includes a
well-bearing component having a plurality of wells disposed on a surface. Suitable
25 devices include but are not limited to all the devices discussed in the introduction
hereinabove such as the well-bearing components taught in U.S. Patent 4,729,949, PCT
patent application US99/04473, PCT patent application IL04/000192 and PCT patent
application IL01/000992. It is important to note that the property of gels to allow cell
proliferation therein or the property of gels to delay cell-proliferation are in some
30 embodiments of secondary importance to the gel cover of the present invention. The
teachings of the present invention concerning a gel cover are applicable and useful not
only due to the influence of the gel on the proliferation of living cells, but also for the
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use of the gel as a cover, preventing cells held in a well-bearing component from
moving or being lost.
The gel cover of the present invention can be implemented to cover a well-
bearing component having wells of generally any size. As the teachings of the present
invention are directed to cellular biology, it is generally preferred that the wells be small
so as to avoid a large number of cells from being held in any one well. Thus, generally,
the dimensions of the wells are generally less than about 200, 100, 50, 25 or even less
than about 10 microns. The exact size of wells of any given well-bearing component is
determined by the type of cells to be studied using the well-bearing component. Since
different types of cells have different sizes, generally a well-bearing component covered
with a gel cover of the present invention will have wells of a size to accommodate one
or more cells of the type to be studied. Most preferred is that a well be of a size so as to
hold no more than one cell of the type to be studied at any one time. Also preferred is
that a well be of a size so as to hold a predetermined number of cells of the type to be
studied.
Types of suitable gels preferred for implementing a gel cover of the present
invention are as discussed hereinabove. In a preferred embodiment, the gel used as a gel
cover is substantially transparent (whether to visible light, ultraviolet light, infrared
radition or some combination thereof). In some embodiments of the present invention it
is desirable to include an active entity in a gel cover of the present invention.
Methods of manufacture of a device of the present invention
With the exception of the carrier, a chip-device of the present invention is
produced using methods with which one skilled in the art is acquainted and described,
for example, in PCT patent application ILO 1/000992.
A carrier of the present invention is produced using any of a variety of methods
known in the art. Suitable methods include methods that employ one or more techniques
including but not limited to casting, embossing, etching, free-form manufacture,
injection-molding, micro etching, micromachining, microplating, molding, spin coating,
lithography or photo-lithography.
The preferred method of producing a carrier of the present invention is the
method of the present invention. The method of the present invention for producing a
carrier is substantially by providing a template having a negative of the features of the
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surface of the carrier. The template is brought in contact with a precursor material, thus
creating the features of the carrier in the precursor material. The features are
subsequently fixed in the precursor material, thus producing the carrier. Depending on
the precursor material, fixing includes, but is not limited to, methods such as heating the
precursor material, cooling the precursor material, curing the precursor material,
polymerizing the precursor material, cross-linking the precursor material, irradiating the
precursor material, illuminating the precursor material, gelling the precursor material,
exposing the precursor material to a fixative and waiting a period of time. By fixative is
meant an agent that causes the precursor material to change to the fixed state and is used
herein as a general term for such materials as fixatives, hardeners, polymerization/
crosslinking / curing initiators, catalysts and agents. It is important to note that in some
cases a precursor material is produced by mixing two or more components which
thereafter change to a fixed state, for example, by simply waiting a period of time.
The template having a negative of the features is, for example, a stamp or a
mold, and is made of any suitable material that is more rigid than a respective precursor
material, including but not limited to elastically deformable materials, plastically
deformable materials, ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, paraffins, polystyrene, polyurethanes, polyvinyl chloride,
silicon, silicon oxide, silicon rubbers and wax.
The template is made, for example, using methods, with which one skilled in the
art is acquainted such as casting, embossing, etching, free-form manufacture, injection-
molding, microetching, micromachining, microplating, molding, lithography or photo-
lithography. The features created in the precursor material by the contact of the template
include the wells and additional features such as drains, channels, coupling elements,
drains, fluid channels, fluid reservoirs (having U-shaped or V-shaped profiles), input
ports, light sources, magnetizable elements, membranes, microreactors, micro valves,
passages, optical components, optical fibers, optical filters, output ports, plumbing
routes, pumps, transport channels, valves, and fiducial points. Features also include
protruberances for separating wells from each other, protruberances for supporting a
cover, protruberances for implementing the methods of the present invention, walls and
partial walls.
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In Figure 4, is shown a reproduction of a scanning electron micrograph of the
domes on a nickel stamp used as a template for the production of a carrier of the present
invention. Seen is an array of hexagonally-packed domes that are the negative of a
hexagonal array of knife-edged wells. The diameter of the domes at the intersection
with the nickel surface is approximately 20 microns.
An important feature created is a feature that is used as a fiducial point, or a
number of features each used as a fiducial point. In one preferred embodiment of the
present invention, a fiducial point is a feature having a special or distinct shape. In a
preferred embodiment of the present invention, when a fiducial point is made according
to the method of the present invention, a marking material {e.g., a fluorescent material
such as fluorescein), a visible material or a metal) is added to the fiducial point,
especially before the features are fixed in the precursor material. The preferred method
of adding a marking material is by applying the material to the respective negative of
the fiducial point on the template. When the template is removed at least some of the
marking material stays in the thus-formed fiducial point.
Once the features are fixed and the carrier produced, the template is separated
from the carrier the carrier cut to size if necessary and the chip-device of the present
invention assembled by attaching the carrier to other chip-device components. Addition
chip-device components include a cover slip, piping, tubing, pumps, fluid supplies,
observation components and the like. In some embodiments, the additional chip-device
components, especially the cover slip, are attached to the carrier using, for example,
adhesives or surface treatments such as anodic bonding, fusion bonding or plasma
treatment such as plasma discharge (exceptionally suitable for polydimethylsiloxane,
see Duffy etal.Anal Chem, 1998, 70,4974-4984).
In one preferred embodiment of the present invention, the precursor material is a
plastically deformable precursor material. Examples of plastically deformable precursor
materials include waxes, paraffins, plastics, polymers and the like. In a preferred
embodiment, the template is a stamp, and the contacting of the template with the
precursor material is substantially stamping the features of the carrier onto the precursor
material, preferably under controlled thermal conditions. In such cases, the precursor
material and the material from which the carrier are generally chemically substantially
similar and there is no need for a separate action to fix the features in the precursor
material beyond separating the produced carrier from the template.
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In another preferred embodiment of the present invention, the precursor material
is an elastic precursor material. Herein by elastic precursor material is meant a material
that is capable of recovering shape after deformation and includes gellable fluids,
polymerizable materials, powders, fluids and thermoplastic materials.
In a preferred embodiment, the elastic precursor material is a thermoplastic
material at an elastic temperature (e.g., when moldable or molten). Subsequent to the
contacting of the template but before the contact is finished, the thermoplastic material
is cooled, thus fixing the desired features in the incipient carrier.
In another preferred embodiment, the elastic precursor material is a
polymerizable material (e.g., monomer solutions, crosslinkable polymers, vulcanizable
polymers, polymerizable fluids, or thermosetting resins). Subsequent to the contacting
of the template but before the contact is finished, the polymerizable material is
polymerized, thus fixing the desired features in the incipient carrier. In such cases, the
precursor material and the material from which the carrier is made are chemically
dissimilar (for example, have the relationship of monomer to polymer).
One preferred polymerizable precursor material is a non-cured
polydimethylsiloxane precursor mixture. A mixture of two polydimethylsiloxane
components (the prepolymer and curing agent) are mixed together in the desired ratio
(preferably about 10:1, but ratios between about 5:1 and about 20:1 are generally
suitable) to give a polydimethylsiloxane precursor mixture, the mixture degassed and
contacted with the template. The features are fixed by the curing of the mixture. Curing
of polydimethylsiloxane precursor generally takes place at room temperature for about
24 hours and, when desired, is accelerated by heating. For example it has been found
that carriers of the present invention made of polydimethylsiloxane are ready for further
processing within 2 hours when cured at 60°C or within 15 minutes when cured at
150°C. A detailed review of methods for the production of micronic features on
polydimethylsiloxane suitable for implementing the teachings of the present invention
are known in the art and discussed, for example, in Ng et aL, Electrophoresis 2002, 23,
3461-3473 and Duffy et aL, Anal. Chem. 1998, 70, 4974-4984.
In Figure 5 is shown a reproduction of a scanning electron micrograph of a well-
matrix of a polydimethylsiloxane carrier manufactured as described herein using the
nickel stamp depicted in Figure 4.
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Another preferred polymerizable precursor material is urethane that is
polymerizable to yield polyurethane.
Another preferred elastic precursor material is a gellable fluid. After the gellable
fluid is brought in contact with the template, the features are fixed by gelling the
gellable fluid to yield a gel Most preferred are gellable fluids that produce a hydrogel.
Methods for gelling gellable fluids known in the art include fluids that gel upon
heating, fluids that gel upon cooling, fluids that gel upon irradiation or illumination,
fluids that gel as a result of contact with a gelling reagent or fluids that gel after a period
of time. Preferred gellable fluids include solutions of proteins, alginates, protein
polysaccharides and low melting temperature agaroses.
One preferred gellable fluid is a low-melting temperature agarose solution. Such
a solution is fluid at temperatures that do not harm living cells (e.g., 20°C) and gel at
low temperatures that do not harm living cells (e.g., 4°C). An exceptionally suitable
agarose for implementing the teachings of the present invention that may be purchased,
for example, from Cambrex Bio Science Rockland Inc. (Rockland, ME, USA) is HGS-
LMP Agarose 0.5% in PBS (catalogue nr. 50221).
Another preferred gellable fluid is an alginate solution which gels upon contact
with a gelling reagent, the preferred gelling reagent being a solution having a Ca" ion
concentration of greater than about 1 x 10~ 6 M. An exceptionally useful gelling agent is
a 20 x 10" 3 NL calcium gluconate solution. Suitable alginate solutions can be purchased
from Pronova Biopolyxners (Drammen, Noway) and include, for example, Protanal
LF120 1% in water and Protanal LF200 1% in water.
A minor technical difficulty occasionally noted during the application of a gel
cover of the present invention is that during the gelling step of the gel cover, the gel
contracts leaving a small gap in the carrier / gel cover interface. It has been found that
such gaps are adequately filled by the addition of additional gellable fluid followed by
gelling of the fluid.
Another preferred method of making a carrier of the present invention is by
photolithography of a spin-coated substrate, a commercially available process (for
example, from Micro Resist Technology GmbH, Berlin, Germany) known to one skilled
in the art. According to such a method, a high aspect ratio photoresist fluid (e.g., SU-8
thich photoresist, MicroChem Corporation, Newton MA, USA) is placed on a planar
substrate (for example a glass slide). The substrate is rotated horizontally, that is, about
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an axis that is perpendicular to the surface of the substrate on which the photoresist fluid
was placed. As a result of the rotation the photoresist fluid forms a uniformly thick layer
on the substrate, typically between about 5 microns and about 20 microns thick.
Subsequently, the photoresist fluid is iUuminated through a mask, fixing only desired
5 areas of the substrate layer. Developing of the substrate with the selectively fixed film
layer removes the non-fixed areas of the film. In such a way a carrier of the present
invention is produced made up of a fixed photoresist layer resting on a substrate layer
where the features of the carrier are carved into the photoresist layer and the bottom of
the features (such as wells) is the substrate. Using photolithography, flat-bottomed wells
10 and other features are easily produced. Such a method is a preferred method of
producing a two-layered carrier of the present invention.
Some embodiments of the present invention include a carrier made of some
material where the wells thereof are coated with a layer that influences the proliferation
of living cells, for example delaying or preventing cell proliferation, for example by
1 5 delaying or preventing adhesion of cells held in the wells.
The material of which a carrier having coated wells according to the teachings of
the present invention is made is any material used in making carriers and includes but is
not limited to elastically deformable materials, plastically deformable materials,
ceramics, epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
20 polydimethylsiloxane, polyethylenterephtalate glycol, polymers, polymethyl
methacrylate, polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon oxide and
silicon rubbers.
One skilled in the art is acquainted with many ways to coat the insides of wells
of a carrier so as to provide a carrier of the present invention having a coating that
25 influences the proliferation of cells held therein.
One preferred method of coating the insides of wells of a carrier of the present
invention, applicable to virtually any carrier produced by virtually any method, is by
vapor deposition. Vapor deposition involves the deposition of materials such as
molecules or atoms onto a surface at low pressures and is characterized by the
30 production of evenly thin, coatings on a surface, such as the surface of a carrier. A
preferred coating for implementing the teachings of the present invention is made of
polymerized para-xylylene molecules (or derivatives thereof) deposited by vapor
deposition, a coating commercially known as Parylene®. Parylene® is preferred not
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only for cell adhesion delaying properties but also for the fact that Parylene® coatings
are uniform, thin (typically 0.1-1 micron) and without voids even when the coated
surface includes configurations with sharp edges, points, flat surfaces, crevices or
exposed internal surfaces.
Experimental methods implented using the device of the present invention
The teachings of the present invention provide the possibility of applying
heretofore difficult or impossible methods for manipulating cells in the field of cellular
biology. Some of the methods are discussed hereinbelow.
A first method of manipulating cells involves holding cells in wells of a well-
bearing component and then covering the held cells with a gel cover. As is discussed in
the hereinabove, there exist many cell-holding well-bearing components. Such
components include the well-bearing components of devices taught in U.S. Patent
4,729,949, PCT patent application US99/04473, PCT patent application IL04/000192
and PCT patent application IL0 1/000992. Preferred, however, is to hold cells in the
wells of a carrier, especially the wells of a carrier of the present invention. For ease of
observation, it is preferred that the well-bearing component be transparent.
Generally, a plurality of cells is held in the wells of a well-bearing component
and a gellable fluid is placed in proximity of the wells, so as to fill the wells without
displacing the cells held therein. It is often covenient to first mix the cells in the gellable
fluid, place the cell/gellable fluid mixture in proximity of the wells and subsequently to
cause the cells to settle into the wells so as to be held in the wells. Settling of cells can
occur simply due to gravity, or can be performed, for example, by centrifugation of the
cells together with the wells. As is discussed hereinabove and in PCT patent application
IL0 1/000992, it is preferred that the wells be juxtaposed. When the wells are justaposed,
the cells settle only in wells and not on the interwell areas.
The gellable fluid is subsequently gelled so as to form a cover. As a result, the
cells are held snugly, without excessive physical stress, between the inside of a
respective well and the surrounding gel cover.
As discussed hereinabove, there exist different types of gellable fluids including
fluids that gel upon heating, fluids that gel upon cooling, fluids that gel upon irradiation
or illumination, fluids that gel as a result of contact with a gelling reagent or fluids that
gel after a period of time. In order to allow study of a cell held in a well according to the
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teachings of the present invention, it is generally preferred that the produced gel cover
be transparent to the appropriate wavelength or wavelengths of light.
It is important to note that in general the teachings of the present invention are
directed to the study of living cells. It is thus necessary that implementation of the
method of the present invention not be lethal or toxic in any significant measure to
living cells.
It is therefore preferred that a gel that is non-toxic and allows transport of
molecules necessary for cell survival and for performing experiment is used for
implementing the teachings of the present invention. Generally hydrogels are non-toxic
and allows transport of molecules such as nutrients, gases, ions and waste to and from a
living cell.
It is also preferred that a gellable fluid that gels under conditions that are
conducive for cell survival be used for implementing the teachings of the present
invention. One preferred gellable fluid is an alginate solution which gels upon contact
with a solution having a Ca 2+ ion concentration of greater than about 1 x 10" 6 M,
concentrations of Ca 2+ ions that are suitable for cells. Another preferred gellable
solution is a solution of low melting temperature agarose. Such solutions are fluid at
relatively low temperatures (e.g., 20°C) and gel at low temperatures that do not harm
living cells {e.g., 4°C).
It is generally preferred that each well hold no more than one cell or no more
than a predetermined number of cells. Most preferred is that the wells be of a size so as
to accommodate no more than one cell. A suspension of cells with a number of cells
greater than the number of wells is placed in proximity of the wells, and the cells
allowed to settle. Excess cells that are "stacked" on top of cells held in wells are
removed, before the gelling of the gellable fluid, for example by the application of a
flow parallel to the top surface of a carrier of a device of the present invention, as
described in PCT patent application IL0 1/000992. In such a manner, substantially all
wells are populated and substantially no cells are left in the area of the matrix and not
held in a well.
In some carriers of the present invention, there are few or no microfluidic
features such as means for producing a flow parallel to the surface of the carrier. For
example, in some embodiments of a carrier the present invention, substantially the only
features are wells and interwell protuberances. In such cases, or for other reasons, it is
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not desired to or it is impossible to produce a flow of fluid parallel to the upper surface
to wash away cells that are riot held in wells. Therefore, an alternative method of
loading a carrier of the present invention that is simpler but in certain instances may be
considered inferior involves adding a suspension of cells in a gellable fluid (preferably a
low temperature liquid agarose) in the proximity of the well matrix of a carrier of the
present invention, where the approximate number of cells in the suspension is
predetermined. The suspension with the carrier is centrifuged, driving the cells into the
wells to be held therein. The gellable fluid is then gelled, for example, by cooling the
the carrier during centrifugation. It has been found that when the number of cells in the
suspension is approximately equal to the number of wells on the carrier, there is
substantially one cell per well, with only minimal stacking of cells on top of already
occupied wells.
In some embodiments of the method of the present invention where a gel cover
is used with a well-bearing component, the wells of the well-bearing component are
individually adressable. As discussed hereinabove when wells are individually
adressable, it is simple to record and identify a cell or cells held in a specific well during
the performance of an experiment. As a result, in such an embodiment a specific cell or
cells can be identified and easily found, even subsequent to moving, transporting,
shipping or storage with no fear that the motion will jostle the held cells out of the
respective wells. For example, the well in which a cell having certain properties is held
is noted. The well-bearing component is moved, for example, to be set in an incubator
or sent to a different laboratory. The cell can thereafter be easily found by reference to
the noted respective individually adressable well.
In some embodiments of the present invention, the bottoms of the wells are
coplanar. When the bottoms of the wells are coplanar, the held cell or cells are easier to
observe without the need for resorting to time-consuming refocussing. This is
exceptionally true when each well holds only one cell. As a result, in such an
embodiment the cells can repeatedly be examined, even subsequent to moving,
transporting, shipping or storage with no fear that the motion will jostle the held cells
out of the respective wells and necessitating time-consuming focussing.
In some embodiments of the present invention, the inside of the wells on which
the cells rest is a proliferation-delaying surface. As discussed hereinabove, a
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proliferation delaying surface is a surface configured to suspend or reduce the rate of
proliferation of cells in contact therewith.
One preferred type of proliferation delaying surface is an adhesion-delaying or
inhibiting surface, that is the surface of the well on which the cell rests is coated with or
made of a material with cell adhesion-delaying or inhibiting properties. As is known to
one skilled in the art, many cell types proliferate only subsequent to adhesion to some
surface or template. In embodiments of the present invention, either the inside of the
wells are coated with an adhesion-delaying material or the well-bearing component
(e.g., a carrier of the present invention) is substantially made of an adhesion-delaying or
inhibiting material.
One class of adhesion-delaying materials includes polydimethylsiloxane. In
accordance with the teachings of the present invention, a polydimethylsiloxane
adhesion-delaying material can include polydimethylsiloxane as one adhesion-delaying
component, can be substantially of polydimethylsiloxane or can be substantially of pure
polydimethylsiloxane. One commercially available polydimethylsiloxane that has been
found to be useful for implementing the teachings of the present invention is RTV 615
(GE Silicones, Wilton, CT, USA).
Another preferred type of proliferation delaying surface is a gel, that is the
surface of the well on which the cell rests is coated with or made of a gel that has
proliferation-delaying properties. In embodiments of the present invention, either the
inside of the wells are coated with a layer of gel or the well-bearing device (e.g., a
carrier of the present invention) is substantially made of a gel, preferably a hydrogel It
is important to note that the gel that is the surface of the wells can be the same, similar
or different as the gel from which the gel cover is made. In such an embodiment,
subsequent to the formation of a gel cover as described hereinabove, the cells held in
wells are in fact encased inside gel, each cell in a respective gel pocket. One
commercially available gel that has been found to be useful for implementing the
teachings of the present invention is a sodium alginate solution marketed under the
name Protanal LF120 1% in water (Pronova Biopolymers, Drammen, Norway).
The use of a gel cover together with a gel adhesion-delaying carrier is discussed
with reference to Figures 6A, 6B and 6C. A glass carrier 12 as described in PCT patent
application BLO 1/00992 including a matrix of hexagonally packed wells, four input ports
and one output port, substantially as described in Figure 1 is provided and held in place
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in rubber frame 42, Figure 6A. Apart from serving to hold glass carrier 12, rubber frame
42 is provided with four input ports 44a, 44b, 44c and 44d 3 and an output port 46 which
are in communication with the inlet connectors 14 and outlet connector 22, respectively
by capillary tubing 30. A rubber polymer 48 (e.g., a hydrophilic vinyl polysiloxane
5 impression material, available as Examix® NDS from GC America Inc., Alsip, IL,
USA) is poured into rubber frame 42 and allowed to harden forming a rubber negative
mold of glass carrier 12. Subsequent to hardening, the rubber negative mold is removed
from rubber frame 42, and glass carrier 12 removed from rubber frame 42. A gellable
fluid (molten agar at 70 °C) is poured into rubber frame 42, and the rubber negative
10 mold put in place in rubber frame 42. After a few hours, the gellable fluid has gelled
and cooled forming a gel carrier. The rubber negative mold is removed and a device of
the present invention assembled using the newly formed gel carrier.
In Figure 6B, depicted in side cross-section is a gel carrier 50 resting on a
transparent holder 24 and held in place by rubber frame 42. A cover slip 52 is held in
15 place above gel carrier 50 by a gasket 54. In such a way, gel carrier 50 and cover slip
52, supported by rubber frame 42 define a sealed volume including a matrix of wells 18,
and fluid flow passages in communication with four flow-generating devices attached to
respective input ports 44a, 44b, 44c and 44d through capillary tubing 30 (represented in
Figure 6B as dashed lines. A suspension of cells in a physiological fluid having a Ca 2+
20 concentration of ImM is injected through input port 44a and is transported through
capillary tubing 30 to the vicinity of matrix of wells 18. The suspended cells are allowed,
to settle into individual wells of matrix of wells 18. Excess cells that have not settled
into a well and physiological fluid are washed away by application of a Ca 2+ -free buffer
solution in a flow parallel to the surface of carrier introduced through input port 44b. A
25 gellable fluid (an alginate) containing calcium gluconate is injected through input port
44c. The concentration of calcium gluconate in the gellable fluid is such that the onset
of gelling takes about 30 minutes after the fluid is injected. After about 30 minutes, the
gellable fluid gels, trapping cells inside a proliferation delaying sandwich.
In Figure 6C, depicted in side cross-section is a gel carrier 50 resting on a
30 transparent holder 24 and held in place by rubber frame 42, where above gel carrier 50
is a gel cover 56 of the present invention.
An exceptionally useful experimental method that is advantageously performed
using the teachings of the present invention involves physically isolating a cell. Once
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cells are snugly held in wells under a gel cover of the present invention, an individual
cell (or cells) is isolated by excising the cell (or cells) from the well-bearing component.
The cell or cells can be further manipulated, encased within the gel, or released by
dissolution of the gel. Clearly excision of cells is most easily accomplished when the
well-bearing component is a carrier of the present invention made of a gel.
It is important to note, that when isolating a cell or cells as described
immediately hereinabove, it is exceptionally useful that the wells be individually
addressable.
In a typical experiment implementing the experimental method described
immediately hereinabove, a carrier of the present invention with a plurality of
fluorescent fiducial points is fashioned from a gel so as to render the wells individually
addressable. A cell-containing fluid is brought in proximity of the wells of the carrier so
that cells in the fluid settle into wells of the carrier to be held therein. A flow of fluid is
applied parallel to the surface of the carrier to wash away cells that are not held in wells.
Subsequently, a gellable fluid is added and gelled. The snugly held cells inside the gel
covered gel carrier are transferred to a microscope and examined. The wells holding
cells having a specific property are noted in reference to the fluorescent fiducial points.
Subsequently, the gel covered gel carrier is transferred to a cutting machine where the
cells having the specific property are excised and separated from the cells not having the
specific property.
An additional useful embodiment of the method of the present invention
includes the addition of active entities with or through the gel cover of the present
invention (or, if applicable, the gel carrier of the present invention). In a first
embodiment, active entities are added subsequent to gelling by contacting an active
entity (generally in solution) with the surface of the gel (be it a gel cover or a gel
carrier). Over the following period of time the active entity diffuses into and through the
gel to the vicinity of the cells held snugly underneath the gel cover. Advantages of this
first embodiment include addition of active entities only when needed. In a second
embodiment, active entities are mixed with the gellable fluid before gelling. Advantages
of the second embodiment include that large active entities that diffuse through a gel
only with difficulty or not at all can be trapped within the gel matrix and that the active
entities are homogenously distributed throughout the gel. Typical active entities useful
in implementing the teachings of the present invention are listed hereinabove.
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In a typical experiment implemeating the experimental method described
immediately hereinabove, a carrier of the present invention is fashioned from a gel
containing a chromatogenic reagent sensitive to a waste product related to cell
metabolism or other secreted compound {e.g., antibodies, enzymes and the like) as a
first active entity. A cell-containing fluid is added above the wells so that cells in the
sample settle into wells of the carrier to be held therein. A flow of fluid is applied
parallel to the carrier to wash away cells that are not held in wells. Subsequently, a
gellable fluid containing a selective toxin configured to kill cells having a specific
mutation as a second active entity is added and gelled. The gel-covered gel carrier is
bathed in a nutrient solution and the development of color by the first active entity is
monitored. Cells having the specific mutation are killed by the action of the second
active entity and do not generate a color. In contrast, cells not having the specific
mutation are easily identified by the generated color.
It is important to note an added advantage of the present invention. Since a gel
matrix reduces the rate of diffusion of compounds secreted from the cell as compared to
regular physiological media and as there is little or no fluid flow inside the gel, it
becomes possible, using the teachings of the present invention, to clearly identify which
cell secretes a given compound, contingent on the existence of an appropriate indicator
or detection method.
As discussed hereinabove, a problem in the art is that of proliferation of cells
held, or isolated in well-bearing devices. A cell is held in a well. If the well is uncovered,
movement of the well-bearing component causes cells to move out from a well, either
being lost or losing identity. Even if the well-bearing component is not moved,
proliferation of cells inside an enclosure leads to unnatural population shapes, cell
distortion and overcrowding effects. Further, if the cell populations grow outwards from
the well, the cells are subject to flow-induced loss or migration from the population
itself. The teachings of the present invention provide a number of solutions for these
problems.
An useful embodiment of the method of the present invention includes allowing
cells snugly held under a gel cover to proliferate into or through the gel cover of the
present invention (or, if applicable, the gel carrier of the present invention).
In a typical experiment implementing the experimental method described
immediately hereinabove, a carrier of the present invention having coplanar wells is
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fashioned from a gel. A cell-containing fluid is added in proximity of the wells so that
cells in the sample settle into wells of the carrier to be held therein, A flow of fluid is
applied parallel to the carrier to wash away cells that are not held in wells.
Subsequently, a gellable fluid containing a chromatogenic active entity is added, the
chromatogenic active entity configured to generate a color only when in contact with a
specific cell-surface receptor. The gel-covered gel carrier is automatically interrogated
with image-processing software using a computer-controlled camera. Cells held in wells
where color is not generated are destroyed by irradiation with a laser. After all
mutation-less cells are destroyed the gel-covered gel carrier is transported to a remote
laboratory. The living cells remain snugly held inside a gel matrix and are not lost. The
proliferation delaying properties of the gel ensure that during transport no additional
cells develop. When arriving at the remote laboratory, the cells are allowed to
proliferate under observation. Since the cells are snugly held, the cells remain coplanar
allowing quick and efficient automatized observation. The cells proliferate into and
through the gel and do not suffer from the effects of overcrowding or limited volume.
Another solution for the problems related to the proliferation of cells held in
prior art a well-bearing device, such as a chip-device, is by holding at least one cell in a
well of a well-bearing device and subsequently increasing the size of the well so as to
provide an increased space for proliferation of the cell. Clearly, a preferred device is a
carrier of a chip-device of the present invention having changeable well sizes, as
described hereinabove. Generally, a plurality of cells is held in wells of the well-bearing
device. The cells are examined (e.g., through visual interrogation, chemical/biological
reaction using an active entity, or a combination of active entities) and certain cells
selected. The non-selected cells are discarded (e.g., by physical removal, for example
using optical tweezers or by damaging (e.g., killing) the non-selected cells). Once the
non-selected cells are discarded, the cell size is increased giving sufficient room for the
cells to proliferate without problems discussed above.
In one embodiment of the present invention a carrier having a changeable well-
size is elastically deformable, for example, the carrier is made substantially of an
elastically deformable material including but not limited to elastomers, rubber, silicon
rubbers or other materials, for example as listed in U.S. Patent 6,740,727, U.S. Patent
6,682,792 and U.S. Patent 6,673,857. Such carriers are generally placed in a deforming
device and by the application of tension stretched to a desired extent. The elastically
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deformable carrier remains in a deforming device and the tension maintained for as long
as the changed size is desired.
The use of an elastically deformable carrier is discussed with reference to
Figures 7A, 7B and 7C. An elastically deformable carrier 58 made of a silicon rubber
5 having a 700% elongation range (e.g., Silastic® LSR 9280-30, Dow Corning
Corporation, Midland, MI, USA) with a matrix of hexagonally packed hexagonal
enclosures 60 is fabricated by press-molding followed by heat curing. Each enclosure
60 has a diameter and a depth of 20 micron. Elastic carrier 58 is placed in a holder /
stretching device 62, Figure 7A. Cells 64 in a cell-containing suspension are allowed to
10 settle into and be held in enclosures 60. Stretching device 62 is used to stretch elastic
carrier 58 so that each of enclosure 60 has a diameter of 100 micron, Figure 7B.
Additionally, a gellable fluid may be applied over the expanded enclosures and gelled as
described above, forming a gel cover that prevents cells 64 held in enclosures 60 from
being lost. Carrier 58, together with the gel cover and stretching device 62 may be
15 moved to an incubator. After some time, cells 64 held in enclosures 60 have
proliferated, Figure 7C.
In another embodiment of the present invention a carrier having a changeable
well-size is plastically deformable, for example, the carrier is made substantially of a
hydrocarbon wax. By plastically deformable material is meant a material that does not
20 recover shape after deformation. Such carriers are generally placed in a deforming
device and by the application of tension stretched to a desired extent. Tension can be
released as the carrier does not recover to the former shape.
The use of a plastically deformable carrier is discussed with reference to Figures
8A, 8B, 8C and 8D. A sheet of hydrocarbon wax (PARAFILM®, Pechiney Plastic
25 Packaging, Inc., Neenah, WI, USA) is placed in a stretching device and pulled to be flat
but with no plastic deformation. A stamp is used to apply a pattern to the surface of the
wax sheet so as to make a matrix of hexagonally packed hexagonal enclosures.
Each enclosure 60 has a diameter and a depth of 20 micron. Plastically
deformable carrier 66 is placed in a holder / stretching device 62, Figure 8 A. Cells 64 in
30 a cell-containing suspension are allowed to settle into and be held in enclosures 60.
Stretching device 62 is used to stretch Plastically deformable carrier 66 so that each of
enclosure 60 has a diameter of 100 micron, Figure 8B. According to one embodiment, a
gellable fluid may be applied over the expanded enclosures and gelled as described
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above, forming a gel cover that prevents cells 64 held in enclosures 60 from being lost.
Plastically deformable carrier 66 is released from stretching device 62 and, together
with the gel cover, is moved to an incubator, Figure 8C. After some time, cells 64 held
in enclosures 60 have proliferated, Figure 8D. When sufficient proliferation occurs,
cells are harvested from each clone for further examination, with the possibility of
leaving at least one cell in the original enclosure.
In a variation of the methods discussed immediately hereinabove, a series of
expandable carriers of the present invention is provided. The size of wells of one carrier
of the series in the expanded state is substantially similar to the size of wells on the
succeeding carrier of the series in the non-expanded state. In such a way, a cell is
isolated in a well of a first carrier. The size of the well of the first carrier is expanded.
The succeeding carrier is laid on top of the first carrier. The two carriers are inverted so
that cells in the expanded wells of the first carrier drop into the the non-expanded wells
of the second carrier. The process is repeated until the cell is deemed to have sufficient
space to proliferate.
Another aspect of the present invention involves a method of collecting cells
from a biological sample (such as but not limited to tumors, organs, flesh, tissues and
tissue samples) by pressing the biological sample onto a well-bearing surface of a well-
bearing component. Preferably the well-bearing surface includes a plurality of cells
configured to hold at least one cell of a certain type (and preferably no more than one)
and also includes a plurality of protuberances protruding from the surface.
When it is desired to collect cells from a biological sample, the biological
sample is laid upon or pressed against the well-bearing surface, releasing whole living
cells from the biological sample. The release of whole living cells increases when prior
to pressing the biological sample on the surface a trauma is applied to the biological
sample. Typical traumas include crushing, cutting, macerating, mashing, slicing,
squishing and squeezing the biological sample.
As is discussed in the hereinabove, there exist many well-bearing components
all of which, with the appropriate modification, are suitable for implementing the
method of collecting cells from a biological sample of the present invention. Such
components include the well-bearing components of devices taught in U.S. Patent
4,729,949, PCT patent application US99/04473, PCT patent application IL04/000192
and PCT patent application IL01/000992. Preferred devices for implementing the
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method of collecting cells of the present invention are devices where the well-bearing
component is a carrier of a chip-device, especially a chip-device having a removeable
cover slip of the present invention or of a chip device of PCT patent application
IL0 1/00992. Also preferred is a well-bearing device of PCT patent application
IL04/000194.
Generally, the protruberances on the surface need to be relatively hard, that is
made of materials such as, but not limited to, plastic, polymer, metals, glass and silicon.
Although sharp protruberances are effective in removing cells from a biological sample,
it has been found that more viable and undamaged cells are harvested when the
protuberances are not sharp. The typical size of protuberances useful for removing cells
from a biological sample in accordance with the teachings of the present invention are
between about 1 micron high and about 20 microns high. "Sharp" is a relative term, and
depends on the dimensions of the cells to be harvested from the biological sample. It has
been found that so as not to pierce a cell under the conditions used but still effectively
assist in removal of the cell from the biological matrix, a "not sharp" protuberance is
generally of a tip-width between about 5% and about 30% of the cell diameter, or
preferably a tip-width of between about 10% and about 20% of the cell diameter. Since
in a typical embodiment of the present invention a well for holding a single cell is of the
dimensions of the cell, it is a simple matter to calculate an acceptable width of a
protuberance for a given carrier of the present invention. For example, a carrier
designed for studying and holding cells having a diameter of about 5 microns,
protuberances will typically have a tip-width of between 0.25 microns (area of ca. 0.5
micron 2 ) and 1.5 microns (area of ca. 1.8 micron 2 ). For example, a carrier designed for
studying and holding cells having a diameter of about 10 microns, protuberances will
typically have a tip-width of between 0.5 microns (area of ca, 0.2 micron 2 ) and 3
microns (area of ca, 7 micron 2 ). For example, a carrier designed for studying and
holding cells having a diameter of about 20 microns, protuberances will typically have a
tip-width of between 1 microns (area of ca. 0.8 micron 2 ) and 6 microns (area of ca. 28
micron 2 ). As is clear to one skilled in the art, the calculation of a suitable tip width is a
simple matter for one skilled in the art. Generally, it is preferred that the protruberances
protrude from the areas between the wells.
In general, cell viability and cell harvesting is improved when the surface and
the wells thereupon are filled with a fluid (e.g., water or a physiological fluid). Cell
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viability and cell harvesting is also improved by providing a flow of a fluid along the
biological sample during the harvesting process.
Once a sufficient time has passed for sufficient cell-harvest, a cover is placed on
or over the surface so as to allow manipulation, analysis and treatment of the harvested
5 cells as is described herein, in PCT patent application ILO 1/000992 or in PCT patent
application IL04/000194.
An typical procedure for collecting cells from a tumor is described with
reference to Figures 10A and 10B. A transparent carrier 68, made in accordance with
the teachings of the present invention or in accordance with the teachings of PCT patent
10 application ILO 1/000992, is provided having a well matrix 18 including a matrix of
hexagonally packed knife-edged hexagonal wells having a plurality of protuberances
emerging from the area between any three wells, similar to the protuberances depicted
in Figures 9A and 9B. Transparent carrier 68 is mounted in a holder 42, where cover
slip 52 is held in place by gasket 54. An Met flow device is in comunication with the
1 5 fluid channels of transparent carrier 69 through capillary tubes 30. The inlet flow device
is activated, filling the fluid channels, reservoirs and wells with fluid and driving air
bubbles out of the system. Figure 10A.
Once the fluid channels of carrier 68 are filled with fluid and devoid of air,
gasket 54 is released and cover slip 52 removed, exposing well matrix 18. A biological
20 sample 70, (e.g., a tumor) is pressed against well matrix 18 while fluid 72 is made to
flow along biological sample 70, Figure 10B. After a few seconds or up to a few
minutes, during which time cells freed from biological sample 70 by the action of the
protuberances of well matrix 18 settle down into wells, biological sample 70 is set aside,
cover slip 52 and gasket 54 returned to the proper position and study of the thus-
25 collected cells performed in the usual way.
As is clear to one skilled in the art, the quick and simple method of harvesting
cells of the present invention allows, for example, high throughput and efficient
screeing of biological samples, for example in the fields of genetics, diagnostics and
oncology.
30 In a typical example, a transparent polydimethylsiloxane carrier of the present
invention with a surface having hexagonally packed individually addressable wells with
protuberances surrounded by a wall is placed in chip-device, A drop of physiological
fluid is placed on the carrier so as to completely fill the wells with the fluid. A tumor is
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excised from a patient and pressed against the surface while more physiological fluid is
dripped along the tumor, A glass cover slip with cover slip electrodes is placed on top of
the carrier so as to rest on the wall of the carrier. As described in PCT patent application
IL0 1/000992, individual cells are held in individual respective wells. A chromatogenic
reagent configured to generate a color upon contact with a pathological cell is
introduced. Cells that do not react with the reagent are extracted by the application of an
attractive force from a cover slip electrode associated with the respective well A
diagnosis is then made based on the reaction with the reagent. When only pathological
cells remain in the well, a gellable fluid is introduced and gelled, trapping the held cells
between the dimethylsiloxane carrier and a gel cover. The carrier and trapped cells are
then stored in non-proliferating conditions for further examination and analysis if
required.
Additional objects, advantages, and novel features of the present invention will
become apparent to one ordinarily skilled in the art upon examination of the following
examples, which are not intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated hereinabove and as
claimed in the claims section below finds experimental support in the following
examples.
EXPERIMENTAL RESULTS
Cell-Proliferation delay of poly dimethylsUoxane (FBM§)
A standard glass petri dish (Nunc S/A, Roskilde, Denmark) and two petri dishes
made of polydimethylsiloxane, Dish 1 using RTV615 PDMS (GE Silicones, Wilton,
CT, USA) and Dish 2 using Sylgard 184 PDMS (Dow Corning Corporation, Midland,
MI, USA) were provided.
Thawed frozen PCS prostate cancer cells (DSMZ GmbH, Braunschweig,
Germany) were cultured at 37°C in RPMI medium with 10% Fetal Calf Serum in each
of the three dishes. The development on the cells was observed for four days (Table
1A). After four days, the cells were relocated to identical dishes under identical
conditions and again cell development was observed for four days (Table IB).
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Table 1A: PCS cell proliferation in glass and PDMS dishes, Days 0-4
Incubation
24 h
48 h
72 h
96h
Glass
Adherence
proliferation
start
proliferation
proliferation
Dish 1
no adherence
adherence
proliferation
start
proliferation
Dish 2
no adherence
adherence
proliferation
start
proliferation
Total time
Table IB: PC3 cell proliferation in glass and PDMS dishes, Days 5-8
Incubation
Glass
Dish 1
Dish 2
120 h
24 h
Adherence
no adherence
no adherence
144 h
48 h
proliferation
start
no adherence
no adherence
168 h
72 h
proliferation
adherence
adherence
192 h
96h
proliferation
proliferation
start
proliferation
start
From the results observed and summarized in Tables 1A and IB, it is seen that
polydimethylsiloxane delays cell adherence to a surface and thus delays-proliferation. It
is important to note that the non-cytotoxicity of cells of polydimethylsiloxane was
confirmed as no cell deaths were observed.
Generally, the nomenclature used herein and the laboratory procedures utilized
in the present invention include techniques from the fields of biology, chemistry and
engineering. Such techniques are thoroughly explained in the literature.
It is appreciated that certain features of the invention, which are, for clarity,
described in the context of separate embodiments, may also be provided in combination
15 in a single embodiment. Conversely, various features of the invention, which are, for
brevity, described in the context of a single embodiment, may also be provided
separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific
20 embodiments thereof, it is evident that many alternatives, modifications and variations
will be apparent to those skilled in the art. Accordingly, the present invention is
intended to embrace all such alternatives, modifications and variations that fall within
the spirit and broad scope of the appended claims. All publications, patents and patent
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applications mentioned in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each individual publication,
patent or patent application was specifically and individually indicated to be
incorporated herein by reference. In addition, citation or identification of any reference
5 in this application shall not be construed as an admission that such reference is available
as prior art to the present invention.
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WHAT IS CLAIMED IS
1. A chip-device for holding living cells, the device comprising a carrier
having a plurality of wells disposed on a surface each well configured to hold at least
one living cell, the device characterized in that said wells are configured to influence the
proliferation of living cells held in said wells.
2. The device of claim 1 5 wherein the inside of said wells comprises a
material selected from the group consisting of a gel, a hydrogel, polydimethylsiloxane,
an elastomer, polymerized para-xylylene molecules, polymerized derivatives of para-
xylylene molecules and silicon rubber.
3. The device of claim 1, wherein said carrier is substantially made of a
material selected from the group consisting of a gel, a hydrogel, polydimethylsiloxane,
an elastomer and silicon rubber.
4. The device of claim 1, wherein said influence is predetermined.
5. The device of claim 1, wherein said configuration is at least one feature
from amongst the six features:
(a) the inside of said wells is configured to delay cell proliferation;
(b) the inside of said wells is configured to inhibit cell proliferation;
(c) said wells are configured to allow cell proliferation inside at least one
component of said chip-device;
(e) the inside of said wells is configured to delay adhesion of living cells thereto;
(f) the inside of said wells is configured to inhibit adhesion of living cells
thereto; and
(g) the size of said wells is changeable.
6.
The device of claim 5, wherein the size of said wells is changeable.
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7. The device of claim 6, said carrier configured to be deformable in at least
one dimension and that upon deformation the size of at least one of said wells is
changed
8. The device of claim 7, said carrier being elastically deformable,
9. The device of claim 8, said carrier substantially made of an elastically
deformable material selected from the group consisting of elastomers, rubber and silicon
rubbers.
10. The device of claim 7, said carrier being plastically deformable.
11. The device of claim 10, said carrier substantially made of a plastically
deformable material selected from the group consisting of hydrocarbon wax, crystalline
wax, polypropylene, isotactic polypropylene homopolymer, syndiotactic polypropylene
homopolymer, metallocene catalyzed isotactic polypropylene homopolymer,
metallocene catalyzed syndiotactic polypropylene homopolymer, ethylene-propylene
random copolymer, butene-propylene random copolymer, ethylene-propylene-butene-1
terpolymer, low density polyethylene, linear low density polyethylene, very low density
polyethylene, metallocene catalyzed polyethylene, metallocene catalyzed polyethylene
copolymers, ethylene-methacrylate copolymers, ethylene-vinyl acetate copolymers,
ionomer resins, an ethylene-propylene random copo^mer, ethylene-butene-1
copolymer, ethylene-propylene-butene-1 terpolymer, propylene-butene copolymer, low
density polyethylene, linear low density polyethylene, very low density polyethylene,
metallocene catalyzed polyethylene plastomer, metallocene catalyzed polyethylene,
metallocene catalyzed polyethylene copolymers, ethylene-methacrylate copolymer,
ethylene vinyl acetate copolymer, ionomer resin and combinations thereof.
12. The device of claim 1, said carrier is substantially of a material having an
index of refraction similar to that of water.
13. The device of claim 12, said carrier is of a material having an index of
refraction less than about 1 A
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14. The device of claim 12, said carrier is of a material having an index of
refraction less than about 1.38.
15. The device of claim 12, said carrier is of a material having an index of
refraction less than about 1.36.
16. The device of claim 12, said carrier is of a material having an index of
refraction less than about 1.35.
17. The device of claim 12, said carrier is of a material having an index of
refraction less than about 1.34.
1 8 . The device of claim 1 , having at least one component made of a gel.
19. The device of claim 18, wherein said gel is substantially transparent.
20. The device of claim 18, wherein said gel is a hydrogel.
21. The device of claim 18, wherein said gel is made of a material selected .
from the group consisting of agar gels, agarose gels, gelatins, low melting temperature
agarose gels, alginate gels, room-temperature Ca 2+ -induced. alginate gels and
polysaccharide gels.
22. The device of claim 18, wherein the water content of said gel is greater
than about 80% by weight.
23. The device of claim 18, wherein the water content of said gel is greater
than about 92% by weight.
24. The device of claim 18, wherein the water content of said gel is greater
than about 95% by weight.
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25. The device of claim 18, wherein the water content of said gel is greater
than about 97% by weight.
26. The device of claim 18, wherein the water content of said gel is greater
than about 98% by weight.
27. The device of claim 18, wherein said gel comprises an active entity.
28. The device of claim 27, wherein said active entity is selected from the
group consisting of antibodies, antigens, biological materials, chemical materials,
chromatogenic compounds, drugs, enzymes, fluorescent probes, immunogenes,
indicators, ligands, nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
29. . The device of claim 18, wherein said carrier is made of said gel.
30. The device of claim 18, wherein a cover for said surface is made of said
gel
31. The device of claim 30 9 wherein said carrier is made of a material
selected from the group consisting of gels, hydrogek, agar gels 5 agarose gels, gelatins,
low melting temperature agarose gels, alginate gels 9 room-temperature Ca 2+ -induced
alginate gels, polysaccharide gels, elastically deformable materials, plastically
deformable materials, ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, paraffins, polystyrene, polyurethanes, polyvinyl chloride,
silicon, silicon oxide, silicon rubbers and wax.
32. The device of claim 30 wherein said carrier is made of a second gel.
33. The device of claim 32, wherein said second gel is substantially the same
as said gel from which said cover is made.
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34. The device of claim 32, wherein said second gel is substantially different
from said gel from which said cover is made.
35. The device of claim 1, wherein the inside of said wells is configured to
delay adhesion of living cells thereto.
36. The device of claim 35, wherein the inside of said well comprises a
material that delays adhesion of living cells thereto.
37. The device of claim 36, wherein said carrier is substantially fashioned
from said adhesion-delaying material.
38. The device of claim 36, wherein the inside of said wells is coated with
said adhesion-delaying material.
39. The device of claim 36, wherein said adhesion-delaying material
comprises polydimethylsiloxane.
40. The device of claim 39, wherein said adhesion-delaying material is
substantially polydimethylsiloxane.
41. The device of claim 39, wherein said adhesion-delaying material is
substantially pure polydimethylsiloxane.
42. The device of claim 1, wherein said wells are juxtaposed.
43. The device of claim 42, the interwell area between two said wells is less
then about 0.35 the sum of the areas of said two wells.
44. The device of claim 42, the interwell area between two said wells is less
then about 0.25 the sum of the areas of said two wells.
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45. The device of claim 42, the interwell area between two said wells is less
then about 0.15 the sum of the areas of said two wells.
46. The device of claim 42, the interwell area between two said wells is less
then about 0.10 the sum of the areas of said two wells.
47. The device of claim 42, the interwell area between two said wells is less
then about 0,06 the sum of the areas of said two wells.
48. The device of claim 42, wherein a rim of a said well is substantially
knife-edged.
49. The device of claim 1, wherein the dimensions of said wells are less than
about 200 microns.
50. The device of claim 1, wherein the dimensions of said wells are less than
about 100 microns.
51. The device of claim 49, wherein the dimensions of said wells are less
than about 50 microns.
52. The device of claim 49, wherein the dimensions of said wells are less
than about 25 microns.
53. The device of claim 49, wherein the dimensions of said wells are less
than about 10 microns.
54. The device of claim 1 , wherein said wells are configured to hold no more
than one living cell of a certain type.
55. The device of claim 1, wherein said wells are configured to hold a
predetermined number of living cells of a certain type.
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56. The device of claim 1, wherein said wells are enclosures of dimensions
such that substantially an entire cell of a certain type is containable within a said
enclosure, each said enclosure having an opening at said surface, said opening defined
by a first cross section of a size allowing passage of a cell of said certain type.
57. The device of claim 56, wherein the volume of said enclosure is less than
about 1 x 10~ n liter.
58. The device of claim 56, wherein the volume of said enclosure is less than
about 1 x 10" 12 liter.
59. The device of claim 56, wherein the volume of said enclosure is less than
about 1 x 10" 13 liter.
60. The device of claim 56, wherein the volume of said enclosure is less than
about 1 x 10" 14 liter.
6 1 . The device of claim 56, wherein the volume of said enclosure is less than
about 1 x 10~ 15 liter.
62. The device of claim 56, wherein the area of said first cross section is less
than about 40000 micron 2 .
63. The device of claim 56, wherein the area of said first cross section is less
than about 10000 micron 2 .
64. The device of claim 56, wherein the area of said first cross section is less
than about 2500 micron 2 .
65. The device of claim 56, wherein the area of said first cross section is less
than about 625 micron 2 .
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66. The device of claim 56, wherein the area of said first cross section is less
than about 100 micron 2 .
67. The device of claim 56, wherein the dimensions of a said enclosure are
such as to contain no more than one said cell of a certain size at any one time.
68. The device of claim 1, farther comprising protuberances protruding from
said surface between two adjacent wells.
69. The device of claim 68, wherein said protuberances are sharp.
70. The device of claim 69, wherein the area of the tip of said protuberances
is less than about 0.05 micron 2 .
7 1 . The device of claim 68, wherein said protuberances are not sharp.
72. The device of claim 71, wherein the area of the tip of said protuberances
is between about 0.05 micron 2 and 28 micron 2 .
73. The device of claim 68, wherein the height of said protuberances is
between 1 micron and 10 microns high,
74. The device of claim 1, further comprising at least one wall protruding
from said surface, said at least one wall circumscribing at least one area of said surface
where the points of the top edge of said wall define a plane.
75. The device of claim 74, further comprising a cover slip configured to rest
on said top edge of said at least one wall so as to define at least one closed volume
including more than one said well.
76. The device of claim 1, further comprising a cover slip, said cover slip
and said carrier configured so as to allow said cover slip to removeably rest above said
surface substantially in parallel to said surface.
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77. The device of claim 76, wherein said configuration of said carrier
comprises at least one wall protruding from said surface, allowing said cover slip to rest
thereupon substantially in parallel to said surface.
78. The device of claim 76, wherein said configuration of said carrier
comprises at least three protrusions protruding from said surface, said protrusions
having substantially the same height, allowing said cover slip to rest thereupon
substantially in parallel to said surface.
79. The device of claim 76, wherein said cover slip and said carrier are
configured so that there exists a limited number of correct cover slip positions wherein
said cover slip is substantially oriented in a specific position when resting above said
surface.
80. A gel carrier, the carrier having a plurality of wells disposed on a surface
each well configured to hold at least one living cell.
81. A polydimethylsiloxane carrier, the carrier having a plurality of wells
disposed on a surface each well configured to hold at least one living cell.
82. A gel cover for wells of a well-bearing component having a plurality of
wells disposed on a surface each well configured to hold at least one living cell
83. A carrier comprising a first layer of a first material resting on top of a
second layer of a second material, the carrier having a plurality of wells disposed on an
upper surface of said first layer each of said plurality of wells configured to hold at least
one living cell, wherein the bottom of said plurality of wells is said second layer.
84. The carrier of claim 83, wherein said second material is selected from the
group consisting of ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, polystyrene, polyurethanes, polyvinyl chloride, silicon and
silicon oxide.
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85. The carrier of claim 83, wherein said first material is a fixed photoresist
material.
86. A carrier comprising a plurality of wells disposed on a surface each well
configured to hold at least one living cell, the carrier characterized in that bottoms of
said wells are flat.
87. The carrier of claim 86, wherein said carrier comprises a feature in
addition to said wells.
88. The carrier of claim 87, wherein said feature is selected from the group
consisting of channels, fluid channels, fluid reservoirs, microreactors, passages,
plumbing routes, protuberances, transport channels and walls.
89. The carrier of claim 87, wherein said feature has a flat bottom.
90. A chip-device for holding living cells, the device comprising a carrier
having a plurality of wells disposed on a surface each well configured to hold at least
one living cell, the device characterized in that said carrier is made of a material having
an index of refraction similar to that of water.
91. The device of claim 90, said carrier is of a material having an index of
refraction less than about 1.4.
92. The device of claim 90, said carrier is of a material having an index of
refraction less than about 1.38.
93. The device of claim 90, said carrier is of a material having an index of
refraction less than about 1.36.
94. The device of claim 90, said carrier is of a material having an index of
refraction less than about 1.35.
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95. The device of claim 90, said carrier is of a material having an index of
refraction less than about 1.34.
96. The device of claim 90, wherein said carrier is made of a gel.
97. The device of claim 96, wherein said gel is substantially transparent.
98. The device of claim 96, wherein said gel is a hydrogel.
99. The device of claim 96, wherein said gel is made of a material selected
from the group consisting of agar gels, agarose gels, gelatins, low melting temperature
agarose gels, alginate gels, room-temperature Ca 2+ -induced alginate gels and
polysaccharide gels.
100. The device of claim 96, wherein the water content of said gel is greater
than about 80% by weight.
101. The device of claim 96, wherein the water content of said gel is greater
than about 92% by weight.
102. The device of claim 96, wherein the water content of said gel is greater
than about 95% by weight.
103. The device of claim 96, wherein the water content of said gel is greater
than about 97% by weight.
104. The device of claim 96, wherein the water content of said gel is greater
than about 98% by weight.
105. A device for holding living cells, the device comprising:
(a) a well-bearing component having a plurality of wells disposed on a surface,
each well configured to hold at least one living cell; and
(b) a cover covering said surface, said cover substantially made of a gel.
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1 06 . The device of claim 1 05 , wherein said gel is substantially transparent.
107. The device of claim 105, wherein said gel is a hydrogel.
108. The device of claim 107, wherein said gel is made of a material selected
from the group consisting of agar gels, agarose gels, gelatins, low melting temperature
agarose gels, alginate gels, room-temperature Ca 2+ -induced alginate gels and
polysaccharide gels.
109. The device of claim 105, wherein said gel comprises an active entity.
110. The device of claim 109, wherein said active entity is selected from the
group consisting of antibodies, antigens, biological materials, chemical materials,
chromatogenic compounds, drugs, enzymes, fluorescent probes, immunogenes,
indicators, ligands, nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
111. The device of claim 105, wherein the water content of said gel is greater
than about 80% by weight.
112. The device of claim 105, wherein the water content of said gel is greater
than about 92% by weight.
113. The device of claim 105, wherein the water content of said gel is greater
than about 95% by weight.
1 14. The device of claim 105, wherein the water content of said gel is greater
than about 97% by weight.
115. The device of claim 105, wherein the water content of said gel is greater
than about 98% by weight.
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116. The device of claim 105, wherein the dimensions of said wells are less
than about 200 microns.
117. The device of claim 105, wherein the dimensions of said wells are less
than about 100 microns.
118. The device of claim 105, wherein the dimensions of said wells are less
than about 50 microns.
119. The device of claim 105, wherein the dimensions of said wells are less
than about 25 microns.
120. The device of claim 105, wherein the dimensions of said wells are less
than about 10 microns.
121. A method of making a chip-device of claim 1 comprising:
(a) providing a template having a negative of features of said surface of said
carrier;
(b) contacting said template with a precursor material so as to create said
features in said precursor material; and
(c) fixing said features in said precursor material so as to fashion said carrier.
122. The method of claim 121, wherein said fixing comprises a methods
selected from heating said precursor material, cooling said precursor material,
polymerizing said precursor material, cross-linking said precursor material, curing said
precursor material, irradiating said precursor material, illuminating said precursor
material, gelling said precursor material, exposing said precursor material to a fixative
and waiting a period of time.
123. The method of claim 121, wherein said template is made of a material
selected from the group consisting of elastically deformable materials, plastically
deformable materials, ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
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polymethyl methacrylate, paraffins, polystyrene, polyurethanes, polyvinyl chloride,
silicon, silicon oxide, silicon rubbers and wax.
124. The method of claim 121, wherein said features are selected from the
group of features consisting of said wells, channels, coupling elements, drains, fluid
channels, fluid reservoirs, input ports, light sources, magnetizable elements,
membranes, microreactors, microvalves, passages, optical components, optical fibers,
optical filters, output ports, plumbing routes, protruberances, pumps, transport channels,
valves, walls and fiducial points.
125. The method of claim 121, wherein one said feature is a fiducial point and
further comprising:
(d) prior to (c), adding a marking material to said fiducial point.
126. The method of claim 125, wherein said marking material is selected from
the group consisting of metals, fluorescent materials and visible materials.
127. The method of claim 125, further comprising:
(d) prior to (c) applying said marking material onto the respective negative of
said fiducial point.
128. The method of claim 121, further comprising:
(d) subsequent to (c), separating said template from said carrier; and
(e) attaching to said carrier an additional chip-device component.
129. The method of claim 128, wherein said additional chip-device
component is selected from the group consisting of cover slips, piping, tubing, pumps,
fluid supplies and observation components.
130. The method of claim 121, wherein said precursor material is a plastically
deformable precursor material and said fixing said features comprises separating said
template from said precursor material.
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131. The method of claim 121, wherein said plastically defonnable precursor
material is selected from the group consisting of wax, paraffins, plastics, thermoset
materials and polymers.
132. The method of claim 121, wherein said precursor material is an elastic
precursor material.
133. The method of claim 121, wherein said elastic precursor material is
selected from the group consisting of gellable fluids, polymerizable materials, powders
and fluids and thermoplastic materials.
134. The method of claim 133, wherein said elastic precursor material is a
thermoplastic material at plastic temperature and wherein fixing said features comprises
cooling said thermoplastic material.
135. The method of claim 132, wherein said elastic precursor material is a
polymerizable material and wherein fixing said features comprises polymerizing said
polymerizable material.
136. The method of claim 135, wherein said polymerizable material is
selected from the group consisting of monomer solutions, crosslinkable polymers,
vulcanizable polymers, polymerizable fluid and thermosetting resins.
137. The method of claim 136, wherein said polymerizable material is a
polydimethylsiloxane precursor mixture and said fixing said features comprises
polymerizing said polydimethylsiloxane precursor mixture so as to produce
polydimethylsiloxane.
138. The method of claim 136, wherein said polymerizable material includes
urethane and said fixing said features comprises polymerizing said urethane to produce
polyurethane.
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139. The method of claim 132, wherein said elastic precursor material is a
gellable fluid and wherein fixing said features comprises gelling said gellable fluid.
140. The method of claim 139, wherein said gelling said gellable fluid
comprises an action selected from the group consisting of heating said gellable fluid,
cooling said gellable fluid, irradiating said gellable fluid, illuminating said gellable
fluid, contacting said gellable fluid with a gelling reagent and waiting a period of time
for said gellable fluid to gel.
141. The method of claim 139, wherein said gellable fluid is selected from
solutions containing a material selected from the group consisting of agars, agaroses,
gelatins, low melting temperature agaroses, alginates, room-temperature Ca 2+ -inducable
alginates and polysaccharides.
142. The method of claim 141, wherein said gellable fluid is an alginate
solution and said gelling said gellable fluid comprises contacting said gellable fluid with
a gelling reagent.
143. The method of claim 142, wherein said gelling reagent comprises Ca 2+
ions.
144. The method of claim 141, wherein said gellable fluid is a low melting
temperature agarose solution and said gelling said gellable fluid comprises cooling said
gellable fluid.
145. A method of making a chip-device of claim 1 comprising:
(a) providing a carrier having a plurality of wells disposed on a surface, each
well configured to hold at least one living cell; and
(b) coating the inside of said wells with a layer of a material configured to
influence proliferation of living cells held in said wells.
146. The method of claim 145, wherein said coating comprises:
(i) applying a precursor fluid to the inside of said wells; and
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(ii) solidifying said precursor fluid so as to form said layer.
147. The method of claim 146, wherein said solidifying comprises a method
selected from heating said precursor fluid, cooling said precursor fluid, polymerizing
said precursor fluid, cross-linking said precursor fluid, curing said precursor fluid,
irradiating said precursor fluid, illuminating said precursor fluid, gelling said precursor
fluid, exposing said precursor fluid to a fixative and waiting a period of time.
148. The method of claim 145, wherein said coating comprises:
(i) depositing a vapor of said material onto the inside of said wells
thereby forming said layer.
149. The method of claim 145, wherein said coating comprises:
(i) depositing a vapor of a precursor material onto the inside of said
wells; and
(ii) solidifying said precursor material thereby forming said layer.
150. The method of claim 149, wherein said solidifying comprises a method
selected from heating said precursor material, cooling said precursor material,
polymerizing said precursor material, cross-linking said precursor material, curing said
precursor material, irradiating said precursor material, illuminating said precursor
material, gelling said precursor material, exposing said precursor material to a fixative
and waiting a period of time.
151. The method of claim 149, wherein said vapor is a vapor of para-xylylene
molecules or derivatives thereof and said layer comprises polymerized said para-
xylylene molecules or derivatives thereof.
152. The method of claim 145, wherein said surface is made of a material
selected from the group consisting of elastically deformable materials, plastically
deformable materials, ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
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polymethyl methacrylate, polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon
oxide and silicon rubbers.
153. A method of manipulating cells, comprising:
(a) providing a plurality of wells of a well-bearing component, each well
configured to hold at least one living cell;
(b) holding a plurality of living cells in a plurality of said wells;
(c) placing a gellable fluid in proximity with said surface so as to. fill said
plurality of wells; and
(d) gelling said gellable fluid so as to form a gel cover.
154. The method of claim 153, wherein said well-bearing component is a
carrier.
155. The method of claim 153, wherein said well-bearing component is a
carrier of claim 1 .
156. The method of claim 155, wherein said carrier is substantially made of a
gel.
157. The method of claim 153, wherein said placing comprises:
(i) placing a plurality of cells in a gellable fluid in the proximity of said
wells; and
(ii) causing said cells to settle into said wells so as to be held in
respective wells.
158. The method of claim 157, wherein said causing said cells to settle
comprises applying a force to said cells.
159. The method of claim 158, wherein said force is gravitation.
160. The method of claim 158, wherein said force is a centrifugal force.
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161. The method of claim 158, wherein said force is a force resulting from the
impact of photons.
162. The method of claim 158, wherein said force is a force resulting from a
pressure wave.
163. The method of claim 153, wherein said gelling said gellable fluid
comprises an action selected from the group consisting of heating said gellable fluid,
cooling said gellable fluid, irradiating said gellable fluid, illuminating said gellable
fluid, contacting said gellable fluid with a gelling reagent and waiting a period of time
for said gellable fluid to gel.
164. The method of claim 163, wherein said gellable fluid is selected so as to
form a hydrogel upon said gelling.
165. The method of claim 163, wherein said gellable fluid is selected so as to
form a transparent gel upon said gelling.
166. The method of claim 153, wherein said gellable fluid is selected from
solutions containing a material selected from the group consisting of agars, agaroses,
gelatins, low melting temperature agaroses, alginates, room-temperature Ca 2+ -inducable
alginates and polysaccharides.
167. The method of claim 153, wherein said gellable fluid is an alginate and
said gelling said gellable fluid comprises contacting said gellable fluid with a gelling
reagent
168. The method of claim 153, wherein said gellable fluid is a low melting
temperature agarose and said gelling said gellable fluid comprises cooling said gellable
fluid,
169. The method of claim 153, wherein said wells are juxtaposed.
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170. The method of claim 153, wherein prior to (d) each of said plurality of
wells holds no more than one cell.
171. The method of claim 153, wherein the inside of said wells is a
proliferation-delaying surface.
1 72. The method of claim 171, wherein the inside of said wells is a gel.
1 73 . The method of claim 1 72, where said gel is a hydrogel.
174. The method of claim 171, wherein said proliferation-delaying surface is
an adhesion-delaying surface.
175. The method of claim 174, wherein said adhesion-delaying surface
comprises polydimethylsiloxane.
176. The method of claim 174, wherein said adhesion-delaying surface is
substantially polydimethylsiloxane.
177. The method of claim 174, wherein said adhesion-delaying surface is
substantially pure polydimethylsil oxane.
1 78 . The method of claim 153, further comprising:
(e) subsequent to (d), isolating at least one cell by excising said at least one said
cell from said well-bearing component.
179. The method of claim 153, wherein said gellable fluid comprises an active
entity.
180. The method of claim 179, wherein said active entity is selected from the
group consisting of antibodies, antigens, biological materials, chemical materials,
chromatogenic compounds, drugs, enzymes, fluorescent probes, immunogenes,
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indicators, ligands, nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
181. The method of claim 153, further comprising :
(e) subsequent to said gelling, contacting an active entity-containing fluid with
said gel cover.
182. The method of claim 181, wherein said active entity is selected from the
group consisting of antibodies, antigens, biological materials, chemical materials,
chromatogenic compounds, drugs* enzymes, fluorescent probes, immunogenes,
indicators, ligands, nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
183. The method of claim 181, further comprising:
(f) waiting a period of time so as to allow said active entity to diffuse through
said gel cover.
184. The method of claim 153, further comprising :
(e) subsequent to (d), allowing said cells to proliferate into said well-bearing
component.
185. The method of claim 153, further comprising:
(e) subsequent to (d), allowing said cells to proliferate into said gel cover.
186. A method of growing cells comprising:
(a) providing a well-bearing device;
(b) holding at least one living cell in a well of said well-bearing device; and
(c) increasing the size of said well so as to provide an increased space for
proliferation of said cell.
187. The method of claim 186, wherein said well-bearing device is a chip
device.
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188. The method of claim 186, wherein said well-bearing device is a chip
device of claim 6.
189. The method of claim 186, further comprising :
(d) prior to (c), holding a plurality of living cells in a plurality of wells of said
well-bearing device; and
(e) increasing the size of said wells so as to provide an increased space for
proliferation of said held living cells.
190. The method of claim 189, wherein during (d) each of said plurality of
wells holds no more than one cell.
191. The method of claim 189, wherein said discarding comprises physically
moving at least one of said non-selected cells.
192. The method of claim 189, wherein said discarding comprises damaging
at least one of said non-selected cells.
193. A method of collecting cells from a biological sample comprising:
(a) providing a well-bearing device, said well -bearing device having:
(i) a plurality of wells disposed on a surface, each well configured
to hold at least one cell; and
(ii) a plurality of protuberances protruding from said surface
(b) contacting the biological sample with said surface so as to remove cells
from the biological sample.
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FIG. 7A
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FIG. 7B
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FIG. 7C
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FIG. 8A
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FIG. 8C
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FIG. 8D
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INTERNATIONAL SEARCH REPORT
Internal nal application No.
PCT/IL04/00571
A. CLASSIFICATION OF SUBJECT MATTER
IPC(7) : C12M 3/00; C12N 5/00
US CL : 435/395, 305.2
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classifxcation system followed by classification symbols)
U.S. : 435/30, 395-397, 401, 402, 288.4, 288.5, 293.1, 297.2, 297.5, 305.2, 305.3, 309.1; 359/398
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
DOCUMENTS CONSIDERED TO BE RELEVANT
Category *
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Y
US 2002/0173033 Al (HAMMERICK et al.) 21 November 2002 (21.11.2002), see entire
1-193
document.
Y
US 5,627,045 A (BOSHNER et al.) 06 May 1997 (06.05.1997), see entire document.
1-193
Y
US 4,308,351 A (LEIGHTON et al.) 29 December 1981 (29.12.1981), see entire document.
1-193
Y
US 2003/0030184 Al (KIM et al.) 13 February 2003 (13,02.2003), see entire document.
1-193
Y
WO 03/035824 Al (DEUTSCH et al.) 01 May 2003 (01.05.2003), see entire document.
1-193
Y
US 6,103,479 A (TAYLOR) 15 August 2000 (15.08.2000), see entire document.
1-193
□
Further documents are listed in the continuation of Box C.
□
See patent family annex.
* Special categories of cited documents: "T"
" A M document defining the general state of the art which is not considered to be
of particular relevance
"X"
W E" earlier application or patent published on or after the international filing date
document which may throw doubts on priority claim(s) or which is cited to
establish the publication date of another citation or other special reason (as "Y"
specified)
"O" document referring to an oral disclosure, use, exhibition or other means
"P M document published prior to the international filing date but later than the U & M
priority date claimed
later document published after the international filing date or priority
date and not in conflict with the application but cited to understand the
principle or theory underlying the invention
document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive step
when the document is taken alone
document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art
document member of the same patent family
Date of the actual completion of the international search
01 November 2004 (01.11.2004)
Date of mailing of the international search report
09 NOV 2004
Name and mailing address of the ISA/US
Mail Stop PCT, Attn: ISA/US
Commissioner for Patents
P.O. Box 1450
Alexandria, Virginia 22313-1450
Facsimile No. (703) 305-3230
Authorized officer
William H. Beisner K iJL^tv^
Telephone No. 571-272-1700 ^ j
Form PCT/ISA/210 (second sheet) (January 2004)
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